Replication-competent anti-cancer vectors

ABSTRACT

Novel vectors which are replication competent in neoplastic cells and which overexpress an adenovirus death protein are disclosed. Some of the disclosed vectors are replication-restricted to neoplastic cells or to neoplastic alveolar type II cells. Compositions and methods for promoting the death of neoplastic cells using these replication-competent vectors are also disclosed.

This application is a continuation of co-pending U.S. patent applicationSer. No. 09/351,778. filed July 12, 1999, the entire content of which isincorporated herein by reference in its entirety.

REFERENCE TO GOVERNMENT GRANT

This invention was made with government support under a grant from theNational Institutes of Health, Grant Number RO1 CA71704. The governmenthas certain rights in this invention.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

This invention relates generally to the treatment of cancer and moreparticularly to vectors which replicate in neoplastic cells and whichoverexpress an adenovirus death protein (ADP) and to the use of thesevectors in treating human cancer.

(2) Description of the Related Art

Cancer is a leading cause of death in the United States and elsewhere.Depending on the type of cancer, it is typically treated with surgery,chemotherapy, and/or radiation. These treatments often fail: surgery maynot remove all the cancer; some cancers are resistant to chemotherapyand radiation therapy; and chemotherapy-resistant tumors frequentlydevelop. New therapies are necessary, to be used alone or in combinationwith classical techniques.

One potential therapy under active investigation is treating tumors withrecombinant viral vectors expressing anti-cancer therapeutic proteins.Adenovirus-based vectors contain several characteristics that make themconceptually appealing for use in treating cancer, as well as fortherapy of genetic disorders. Adenoviruses (hereinafter usedinterchangeably with “Ads”) can easily be grown in culture to high titerstocks that are stable. They have a broad host range, replicating inmost human cancer cell types. Their genome can be manipulated bysite-directed mutation and insertion of foreign genes expressed fromforeign promoters.

The adenovirion consists of a DNA-protein core within a protein capsid(reviewed by Stewart et al., “Adenovirus structure by x-raycrystallography and electron microscopy.” in: The Molecular Repertoireof Adenoviruses, Doerfler, W. et al., (ed)., Springer-Verlag,Heidelberg, Germany, p. 25-38). Virions bind to a specific cellularreceptor, are endocytosed, and the genome is extruded from endosomes andtransported to the nucleus. The genome is a linear duplex DNA of about36 kbp, encoding about 36 genes (FIG. 1A). In the nucleus, the“immediate early” E1A proteins are expressed initially, and theseproteins induce expression of the “delayed early” proteins encoded bythe E1B, E2, E3, and E4 transcription units (reviewed by Shenk, T.“Adenoviridae: the viruses and their replication” in: Fields Virology,Field, B. N. et al., Lippencott-Raven, Philadelphia, p. 2111-2148). E1Aproteins also induce or repress cellular genes, resulting in stimulationof the cell cycle. About 23 early proteins function to usurp the celland initiate viral DNA replication. Viral DNA replicates at about 7 hpost-infection (p.i.), then late genes are expressed from the “majorlate” transcription unit. Major late mRNAs are synthesized from thecommon “major late promoter” by alternative pre-mRNA processing. Eachlate mRNA contains a common “tripartite leader” at its 5′-terminus(exons 1, 2, and 3 in FIG. 1), which allows for efficient translation ofAd late mRNAs. Cellular protein synthesis is shut off, and the cellbecomes a factory for making viral proteins. Virions assemble in thenucleus at about 1 day p.i., and after 2-3 days the cell lyses andreleases progeny virus. Cell lysis is mediated by the E3 11.6K protein,which has been renamed “adenovirus death protein” (ADP) (Tollefson etal., J. Virol. 70:2296-2306, 1996; Tollefson et al., Virol. 220:152-162,1996). The term ADP as used herein in a generic sense referscollectively to ADP's from adenoviruses such as, e.g. Ad type 1 (Ad 1),Ad type 2 (Ad2), Ad type 5 (Ad5) or Ad type 6 (Ad6) all of which expresshomologous ADP's with a high degree of sequence similarity.

The Ad vectors being investigated for use in anti-cancer and genetherapy are based on recombinant Ad's that are eitherreplication-defective or replication-competent. Typicalreplication-defective Ad vectors lack the E1A and E1B genes(collectively known as E1) and contain in their place an expressioncassette consisting of a promoter and pre-mRNA processing signals whichdrive expression of a foreign gene. These vectors are unable toreplicate because they lack the E1A genes required to induce Ad geneexpression and DNA replication. In addition, the E3 genes are usuallydeleted because they are not essential for virus replication in culturedcells.

A number of investigators have constructed replication-defective Advectors expressing anti-cancer therapeutic proteins. Usually, thesevectors have been tested by direct injection of human tumors growing inmouse models. Most commonly, these vectors express the thymidine kinasegene from herpes simplex virus, and the mice are treated withgancyclovir to kill cells transduced by the vector (see e.g., Felzmannet al., Gene Ther. 4:1322-1329, 1997). Another suicide gene therapyapproach involves injecting tumors with a replication defective Advector expressing cytosine deaminase, followed by administration of5-fluorocytosine (Topf et al., Gene Ther. 5::507-513, 1998).Investigators have also prepared and tested replication-defective Advectors expressing a cytokine-such as IL-2, IL-12, IL-6, tumor necrosisfactor (TNF), type I interferons, or the co-stimulatory molecule B7-1 inthe anticipation that the Ad-expressed cytokine will stimulate an immuneresponse, including cytotoxic T-lymphocytes (CTL), against the tumor(Felzmann et al., supra; Putzer et al., Proc. Natl. Acad. Sci. USA94:10889-10894, 1997). Other vectors express tumor antigens (e.g.melanoma MART1), proteins that de-regulate the cell cycle and induceapoptosis (p53, pRB, p21^(KiP1/WAF1), p16^(CDKN2), and even Ad E1A), andribozymes. An Ad vector expressing FasL induces apoptosis and tumorregression of a mouse tumor (Arai et al., Proc. Natl. Acad. Sci. USA94:13862-13867, 1997).

Despite these generally positive reports, it is recognized in the artthat replication-defective Ad vectors have several characteristics thatmake them suboptimal for use in therapy. For example, production ofreplication-defective vectors requires that they be grown on acomplementing cell line that provides the E1A proteins in trans. Suchcell lines are fastidious, and generation of virus stocks istime-consuming and expensive. In addition, although many foreignproteins have been expressed from such vectors, the level of expressionis low compared to Ad late proteins.

To address these problems, several groups have proposed usingreplication-competent Ad vectors for therapeutic use.Replication-competent vectors retain Ad genes essential for replicationand thus do not require complementing cell lines to replicate.Replication-competent Ad vectors lyse cells as a natural part of thelife cycle of the vector. Another advantage of replication-competent Advectors occurs when the vector is engineered to encode and express aforeign protein. Such vectors would be expected to greatly amplifysynthesis of the encoded protein in vivo as the vector replicates. Foruse as anti-cancer agents, replication-competent viral vectors wouldtheoretically also be advantageous in that they should replicate andspread throughout the tumor, not just in the initial infected cells asis the case with replication-defective vectors.

Wyeth Laboratories developed replication-competent Ad vectors forvaccination purposes, using vaccine strains of Ad serotypes 4, 7, and 5(Lubeck et al., AIDS Res. Hum. Retroviruses 10:1443-1449, 1994). Foreigngenes were inserted into the E3 region (with the E3 genes deleted) orinto a site at the right end of the genome. Two foreign genes used werehepatitis B surface antigen and the HIV envelope protein. They obtainedgood expression in culture, and were able to raise antisera in animalmodels. Phase I human trials were ambiguous, and the project was mostlyabandoned.

Onyx Pharmaceuticals recently reported on adenovirus-based anti-cancervectors which are replication deficient in non-neoplastic cells butwhich exhibit a replication phenotype in neoplastic cells lackingfunctional p53 and/or retinoblastoma (pRB) tumor suppressor proteins(U.S. Pat. No. 5,677,178; Heise et al., Nature Med. 6:639-645, 1997;Bischoff et al., Science 274:373-376, 1996). This phenotype isreportedly accomplished by using recombinant adenoviruses containing amutation in the E1B region that make the encoded E1B-55K proteinincapable of binding to p53 and/or a mutation(s) in the E1A region whichmake the encoded E1A protein (p289R or p243R) incapable of binding topRB and/or the cellular 300 kD polypeptide and/or the 107 kDpolypeptide. E1B-55K has at least two independent functions: it bindsand inactivates the tumor suppressor protein p53, and it is required forefficient transport of Ad mRNA from the nucleus. Because these E1B andE1A viral proteins are involved in forcing cells into S-phase, which isrequired for replication of adenovirus DNA, and because the p53 and pRBproteins block cell cycle progression, the recombinant adenovirusvectors described by Onyx should replicate in cells defective in p53and/or pRB, which is the case for many cancer cells, but not in cellswith wild-type p53 and/or pRB. Onyx has reported that replication of anadenovirus lacking E1B-55K, which is named ONYX-015, was restricted top53-minus cancer cell lines (Bischoff et al., supra), and that ONYX-015slowed the growth or caused regression of a p53-minus human tumorgrowing in nude mice (Heise et al., supra). Others have challenged theOnyx report claiming that replication of ONYX-015 is independent of p53genotype and occurs efficiently in some primary cultured human cells(Harada and Berk, J. Virol 73:5333-5344, 1999). ONYX-015 does notreplicate as well as wild-type adenovirus because E1B-55K is notavailable to facilitate viral mRNA transport from the nucleus. Also,ONYX-015 expresses less ADP than wild-type virus (see Example 1 below).

As an extension of the ONYX-015 concept, a replication-competentadenovirus vector was designed that has the gene for E1B-55K replacedwith the herpes simplex virus thymidine kinase gene (Wilder et al., GeneTherapy 6:57-62, 1999). The group that constructed this vector reportedthat the combination of the vector plus gancyclovir showed a therapeuticeffect on a human colon cancer in a nude mouse model (Wilder et al.,Cancer Res. 59:410-413, 1999). However, this vector lacks the gene forADP, and accordingly, the vector will lyse cells and spread fromcell-to-cell less efficiently than an equivalent vector that expressesADP. The gene for ADP is also lacking in another replication-competentadenovirus vector that has been described, in which a minimalenhancer/promoter of the human prostate specific antigen was insertedinto the adenovirus E1A enhancer/promoter (Rodriguez et al., Cancer Res.57:2559-2563, 1997).

Thus, there is a continuing need for vectors that replicate and spreadefficiently in tumors but that can be modified such that they replicatepoorly or not at all in normal tissue.

SUMMARY OF THE INVENTION

Briefly, therefore, the present invention is directed to novel vectorswhich are replication competent in neoplastic cells and whichoverexpress an adenovirus death protein (ADP). The work reported hereindemonstrates the discovery that overexpression of ADP by a recombinantadenovirus allows the construction of a replication-competent adenovirusthat kills neoplastic cells and spreads from cell-to-cell at a ratesimilar to or faster than that exhibited by adenoviruses expressingwild-type levels of ADP, even when the recombinant adenovirus contains amutation that would otherwise reduce its replication rate innon-neoplastic cells. This discovery was unexpected because it could nothave been predicted from what was known about adenovirus biology that Advectors overexpressing ADP remain viable and that the infected cells arenot killed by the higher amounts of ADP before the Ad vector producesnew virus particles that can spread to other tumor cells. Indeed,naturally-occurring adenoviruses express ADP in low amounts from the E3promoter at early stages of infection, and begin to make ADP in largeamounts only at 24-30 h p.i., once virions have been assembled in thecell nucleus. It is believed that other non-adenoviral vectors can beused to deliver ADP's cell-killing activity to neoplastic cells,including other viral vectors and plasmid expression vectors.

Thus, in one preferred embodiment, the ADP-expressing vector comprises arecombinant adenovirus lacking expression of at least one E3 proteinselected from the group consisting of: gp19K; RIDα (also known as10.4K); RIDβ (also known as 14.5K) and 14.7K. Because these E3 proteinsinhibit immune-mediated inflammation and/or apoptosis of Ad-infectedcells, it is believed that a recombinant adenovirus lacking one or moreof these E3 proteins will stimulate infiltration of inflammatory andimmune cells into a tumor treated with the adenovirus and that this hostimmune response will aid in destruction of the tumor as well as tumorsthat have metastasized. The ADP expressed by preferred embodimentscomprises a naturally-occurring amino acid sequence from a humanadenovirus of subgroup C, namely Ad1, Ad2, Ad5 and Ad6.

In another embodiment, replication of the vector is restricted toneoplastic cells. Such replication-restricted vectors are useful intreating cancer patients in which it is desirable to eliminate or reducedamage to normal cells and tissues that might be caused by the vector,particularly viral vectors that kill the host cell as part of their lifecycle. In preferred embodiments, a recombinant adenovirus has areplication-restricted phenotype because the recombinant adenovirus isincapable of expressing an E1A viral protein which binds the pRB and thep300/CBP proteins or because the E4 promoter has been substituted with apromoter that is activated only in neoplastic cells.

In yet another embodiment, the invention provides a vector whichoverexpresses ADP and whose replication is under the control of a tissuespecific promoter or an inducible promoter. In preferred embodiments,the vector comprises a recombinant adenovirus in which the tissuespecific promoter or inducible promoter is substituted for the E4promoter. Such vectors are useful for restricting replication of thevector and its ADP-mediated cell killing to cells of a particular typeor to cells exposed to an exogenous agent that activates the promoter. Apreferred tissue-specific or inducible vector also expresses a phenotypethat restricts its replication to neoplastic cells.

In yet another embodiment, the invention provides a vector whichoverexpresses ADP but which is not restricted to tumors by a specificgenetic modification. Such a vector is more destructive to neoplasticcells than even the naturally occurring Ad's of subgroup C. In preferredembodiments, this vector could be used for patients with terminal cancernot treatable by another method, and who have pre-existing neutralizingantibodies to Ad or to which neutralizing antibodies can beadministered.

In still another embodiment, the invention provides a compositioncomprising a first recombinant virus which is replication competent in aneoplastic cell and overexpresses the adenovirus death protein. In oneembodiment, the recombinant virus is contained within a delivery vehiclecomprising a targeting moiety that limits delivery of the virus to cellsof a certain type. With this embodiment, the replication-competentvector can be of any ADP-overexpressing configuration described herein.In some embodiments, the composition also comprises a second recombinantvirus which is replication-defective and which expresses an anti-cancergene product. The recombinant virus complements spread of thereplication-defective virus, as well as its encoded anti-cancer product,throughout a tumor. In preferred embodiments, the first recombinantvirus is a recombinant adenovirus whose replication is restricted toneoplastic cells and/or which lacks expression of one or more of the E3gp19K; RIDα; RIDβ; and 14.7K proteins.

The ADP-expressing vectors and compositions of the invention are usefulin a method for promoting death of a neoplastic cell. The methodcomprises contacting the neoplastic cell with a vector which isreplication-competent in the neoplastic cell and which overexpressesADP. Where the neoplastic cell comprises a tumor in a patient, thevector is administered directly to the tumor or, in other embodiments,the vector is administered to the patient systemically or in a deliveryvehicle containing a targeting moiety that directs delivery of thevector to the tumor. In embodiments where the vector is a recombinantvirus, the method can also comprise passively immunizing the patientagainst the virus.

In yet another embodiment of the invention, the vector may be used incombination with radiation therapy. The radiation therapy can be anyform of radiation therapy used in the art such as for example, externalbeam radiation such as x-ray treatment, radiation delivered by insertionof radioactive materials within the body near or at the tumor site suchas treatment with gamma ray emitting radionuclides, particle beamtherapy which utilizes neutrons or charged particles and the like. Inaddition, this embodiment encompasses the use of more than one of thevectors of the present invention in a cocktail in combination withradiation therapy.

Another embodiment of the invention involves the use of the recombinantvector in combination with chemotherapy as has been disclosed for otheradenovirus vectors (U.S. Pat. No. 5,846,945). Chemotherapeutic agentsare known in the art and include antimetabolites includingpyrimidine-analogue and purine-analogue antimetabolies, plant alkaloids,antitumor antibiotics, alkylating agents and the like. The use of morethan one of the vectors of the present invention with a chemotherapeuticagent or agents is also contemplated within this embodiment.

Among the several advantages found to be achieved by the presentinvention, therefore, may be noted the provision ofreplication-competent vectors, particularly viruses, which rapidly killcancer cells and spread from cell-to-cell in a tumor; the provision ofsuch vectors whose replication can be induced or which is restricted totumors and/or to cells of a certain tissue type; and the provision ofcompositions and methods for anti-cancer therapy which cause little tono side effects in normal tissues.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of gene expression in Ad5 (FIG. 1A) and KD3, apreferred embodiment of the invention (FIG. 1B), in which the respectivegenomes are represented by the stippled bars and transcription unitsrepresented by arrows above and below the bars, with the E3 proteinslisted above the arrows for the E3 transcription unit, and the L1 to L5families of late mRNA's indicated.

FIG. 2 illustrates the overexpression of ADP by KD1, KD3 , GZ1, and GZ3showing an immunoblot of proteins isolated from human A549 cellsinfected with the indicated viruses and probed with an anti-ADPantibody, with ADP indicating differently glycosylated andproteolytically processed forms of ADP.

FIG. 3 illustrates that the E1A dl1101/1107 mutation referred to in thefigure and hereinafter as dl01/07, retards expression of late proteins,showing an immunoblot of E1A proteins and late proteins in A549 cellsinfected with the indicated viruses in the absence (FIGS. 3A and 3B) orpresence (FIGS. 3C and 3D) of dl327, which has a wild-type E1A regionand has a deletion of all E3 genes but the gene encoding the 12.5Kprotein (FIGS. 3C and 3D). An antiserum specific to the E1A proteins wasused for FIGS. 3A and 3C. An antiserum raised against Ad5 virions wasused for FIGS. 3B and 3D.

FIG. 4 illustrates that KD1 and KD3 kill cells more efficiently thancontrol viruses that express less or no ADP, showing a graph of thepercent of A549 cells infected with the indicated viruses that wereviable at the indicated days p.i. as determined by trypan blueexclusion.

FIG. 5 is a cell spread assay illustrating that overexpression of ADPenhances spread of virus from cell to cell, showing monolayers infectedwith the indicated viruses at the indicated PFU/cell which were treatedat 7 days p.i. with crystal violet, which stains live cells but not deadcells.

FIG. 6 illustrates that KD1 and KD3 replicate well in growing cells butnot in growth-arrested cells showing the virus titer extracted fromgrowing or growth arrested HEL-229 cells at various times followinginfection with 100 PFU/ml of the following viruses: dl309 (FIG. 6A),dl01/07 (FIG. 6B), KD1 (FIG. 6C) and KD3 (FIG. 6D).

FIG. 7 illustrates that KD1 and KD3 are defective in killing primaryhuman bronchial epithelial cells showing these cell monolayers infectedat 30% confluency with 10 PFU/ml of the indicated viruses and stained at5 days p.i. with neutral red.

FIG. 8 illustrates that KD1 and KD3 reduce the growth rate of human A549cell tumors growing in nude mice, showing in FIG. 8A a graph ofaverage-fold increase in tumor size plotted against the number of weeksfollowing infection of the tumor with buffer or with 5×10⁷ PFU at weeklyintervals of or the indicated viruses, and showing in FIG. 8B a similargraph of tumors injected once with 5×10⁸ PFU of KD3 or GZ3.

FIG. 9 illustrates that KD1 and KD3 reduce the growth rate of humanHep3B cell tumors growing in nude mice, showing a graph of average-foldincrease in tumor size plotted against the number of weeks followinginjection of the tumor with buffer or with 5×10⁷ PFU of dl309, KD1 orKD3 at twice weekly intervals of the indicated viruses.

FIG. 10 illustrates that KD1 and KD3 complement the replication andspread of Ad-β-gal, a replication-defective vector that expressesβ-galactosidase, using an infectious center assay showing in FIG. 10A apicture of A549 cell monolayers seeded with A549 cells infected withAd-β-gal alone or with the indicated viruses, with FIGS. 10B and 10Cshowing close-up views of two of the monolayers of FIG. 10A.

FIG. 11 is a bar graph illustrating that KD1 and KD3 increase theexpression of luciferase in human Hep3B cell tumors growing in nudemice, using an assay in which tumors were injected with the indicatedcombinations of viruses, then were extracted 2 weeks p.i. and assayedfor luciferase activity. The numbers in parentheses indicated the foldincrease in luciferase activity compared to that of the Adluc vectorplus buffer.

FIG. 12 is a graph showing the results of a standard plaque developmentassay for KD1 and KD1-SPB on A549 cells engineered to express the TTF1transcription factor (A549/TTF1) and the parental 549 cells, in whichdata are plotted as the number of plaques observed on a particular dayin the assay divided by the final number of plaques observed for thatvirus multiplied by 100.

FIG. 13 is a cell spread assay for KD1 and KD1-SPB on H441 cells andHep3B cells, where cells were infected with the indicated amounts of KD1or KD1-SPB and H441 cells and Hep3B cells were strained with crystalviolet at 5 days p.i. and 8 days p.i., respectively.

FIG. 14 is a graph showing the results of a standard plaque developmentassay for dl309 and two preferred embodiments of the invention, GZ1 andGZ3, in which data are plotted as the number of plaques observed on aparticular day in the assay divided by the final number of plaquesobserved for that virus multiplied by 100.

FIG. 15 is a cell spread assay illustrating that the combination of KD1,KD3, GZ1, or GZ3 with x-ray radiation is more effective in destroyingA549 cell monolayers than is virus vector alone or radiation alone,wherein cells were infected with the indicated amounts of the indicatedviruses, radiated with 600 centigreys (cGy) of x-radiation (bottompanel), or mock radiated (top panel), then stained with crystal violetat 6 days p.i.

FIG. 16 is a graph of a cell spread assay illustrating that 10⁻³ PFU ofKD1, KD3, GZ1, or GZ3 used in combination with 150, 300, or 600centigreys of radiation is more effective in destroying A549 cellmonolayers than virus vector alone or radiation alone. Cell viability isbased on the amount of crystal violet extracted from the culture wells,using the mock-infected non-radiated well as 100% viability.

FIG. 17 illustrates that the combination of KD3 or GZ3 plus x-rayradiation is more effective in reducing the growth of A549 cell tumorsgrowing in nude mice than KD3 alone or GZ3 alone.

FIG. 18 illustrates a structure-function analysis of ADP, showing inFIG. 18A the amino acid sequence of the adenovirus death protein encodedby Ad2 (SEQ ID NO:6), with the various putative domains andglycosylation sites labeled and showing in FIG. 18B a schematic of theADP gene in rec700 and in the indicated deletion mutants, with the rightcolumn summarizing the death promoting phenotype of the various mutantsas a percentage of the wild-type phenotype.

FIGS. 19A and 19B illustrate a cell viability assay of the indicated ADPmutants showing a graph of viability as determined by trypan blueexclusion plotted against hours (FIG. 19A) or days (FIG. 19B)postinfection.

FIG. 20 depicts the amino acid sequence, shown in single letter code,for the ADP proteins of Ad1, Ad2, Ad5, and Ad6 (SEQ ID NOS:5-8), for theAd2 ADP mutants dl716, dl715, dl714, and dl737 (SEQ ID NOS:9-12), andfor putative lumenal Domain (SEQ ID NO:17), transmembrane domain (SEQ IDNO:18), the cytosolic basic-proline domain (SEQ ID NO:19), and theremainder of the cystosolic domain (SEQ ID NO:20) of the ADP protein ofAd2.

FIG. 21 presents the complete nucleotide sequence of the genome of Ad5(SEQ ID NO:28).

FIG. 22 presents the complete nucleotide sequence of the genome of KD1(SEQ ID NO:1).

FIG. 23 presents the complete nucleotide sequence of the genome of KD3(SEQ ID NO:2).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention, it has been discovered thatoverexpression of ADP by a recombinant adenovirus results in fasterlysis of cells and spread of the virus throughout a cell monolayer thanviruses expressing wild-type levels of ADP. It has also been discoveredthat this function for ADP is manifest in an adenovirus which containsE1A mutations that restrict adenoviral replication to neoplastic cells.Thus, vectors which are both replication competent in neoplastic cellsand which overexpress ADP should be useful in anti-cancer therapy.

In the context of this disclosure, the following terms will be definedas follows unless otherwise indicated:

“Naturally-occurring” as applied to an object such as a polynucleotide,polypeptide, or virus means that the object can be isolated from asource in nature and has not been intentionally modified by a human.

“Neoplastic cell” means a cell which exhibits an aberrant growthphenotype characterized by a significant loss of control of cellproliferation and includes actively replicating cells as well as cellsin a temporary non-replicative resting state (G₁ or G₂). A neoplasticcell may have a well-differentiated phenotype or a poorly-differentiatedphenotype and may comprise a benign neoplasm or a malignant neoplasm.

“Recombinant virus” means any viral genome or virion which is differentthan a wild-type virus due to a deletion, insertion, or substitution ofone or more nucleotides in the wild-type viral genome. The recombinantvirus can have changes in the number of amino acid sequences encoded andexpressed or in the amount or activity of proteins expressed by thevirus. In particular, the term includes recombinant viruses generated bythe intervention of a human.

“Replication-competent” as applied to a vector means that the vector iscapable of replicating in normal and/or neoplastic cells. As applied toa recombinant virus, “replication-competent” means that the virusexhibits the following phenotypic characteristics in normal and/orneoplastic cells: cell infection; replication of the viral genome; andproduction and release of new virus particles; although one or more ofthese characteristics need not occur at the same rate as they occur inthe same cell type infected by a wild-type virus, and may occur at afaster or slower rate. Where the recombinant virus is derived from avirus such as adenovirus that lyses the cell as part of its life cycle,it is preferred that at least 5 to 25% of the cells in a cell culturemonolayer are dead 5 days after infection. Preferably, areplication-competent virus infects and lyses at least 25 to 50%, morepreferably at least 75%, and most preferably at least 90% of the cellsof the monolayer by 5 days post infection (p.i.).

“Replication-defective” as applied to a recombinant virus means thevirus is incapable of or is greatly compromised in, replicating itsgenome in any cell type in the absence of a complementingreplication-competent virus. Exceptions to this are cell lines such as293 cells that have been engineered to express adenovirus E1A and E1Bproteins.

“Replication-restricted” as applied to a vector of the invention meansthe vector replicates better in a dividing cell, i.e. either aneoplastic cell or a non-neoplastic, dividing cell, than in a cell ofthe same type that is not neoplastic and/or not dividing, which is alsoreferenced herein as a normal, non-dividing cell. Preferably, areplication-restricted virus kills at least 10% more neoplastic cellsthan normal, non-dividing cells in cell culture monolayers of the samesize, as measured by the number of cells showing cytopathic effects(CPE) at 5 days p.i. More preferably, between 25% and 50%, and even morepreferably, between 50% and 75% more neoplastic than normal cells arekilled by a replication-restricted virus. Most preferably, areplication-restricted adenovirus kills between 75% and 100% moreneoplastic than normal cells in equal sized monolayers by 5 days p.i.

In one embodiment the invention provides a vector that isreplication-competent in neoplastic cells and which overexpresses anADP. Vectors useful in the invention include but are not limited toplasmid-expression vectors, bacterial vectors such as Salmonella speciesthat are able to invade and survive in a number of different cell types,vectors derived from DNA viruses such as human and non-humanadenoviruses, adenovirus associated viruses (AAVs), poxviruses,herpesviruses, and vectors derived from RNA viruses such as retrovirusesand alphaviruses. Preferred vectors include recombinant virusesengineered to overexpress an ADP. Recombinant adenoviruses areparticularly preferred for use as the vector, especially vectors derivedfrom Ad1, Ad2, Ad5 or Ad6.

Vectors according to the invention overexpress ADP. As applied torecombinant Ad and AAV vectors, the term “overexpresses ADP” means thatmore ADP molecules are made per viral genome present in a dividing cellinfected by the vector than expressed by any previously knownrecombinant adenoviral vector or AAV in a dividing cell of the sametype. As applied to other, non-adenoviral vectors, “overexpresses ADP”means that the virus expresses sufficient ADP to lyse a cell containingthe vector.

Vectors overexpressing ADP can be prepared using routine methodology.(See, e.g., A Laboratory Cloning Manual, 2nd Ed., vol. 3, Sambrook etal., eds., Cold Spring Harbor Laboratory Press, 1989). For example, apolynucleotide encoding the ADP can be cloned into a plasmid expressionvector known to efficiently express heterologous proteins in mammaliancells. The polynucleotide should also include appropriate terminationand polyadenylation signals. Enhancer elements may also be added to theplasmid to increase the amount of ADP expression. Viral vectorsoverexpressing ADP can be prepared using similar materials andtechniques.

Where the virus is a recombinant adenovirus, overexpression of ADP canbe achieved in a multitude of ways. In general, any type of deletion inthe E3 region that removes a splice site for any of the E3 mRNAs willlead to overexpression of the mRNA for ADP, inasmuch as more of the E3pre-mRNA molecules will be processed into the mRNA for ADP. This isexemplified in the KD1, KD3, GZ1 and GZ3 vectors (SEQ ID NOS:1-4) whoseconstruction is described below. Other means of achieving overexpressionof ADP in Ad vectors include, but are not limited to: insertion ofpre-mRNA splicing and cleavage/polyadenylation signals at sites flankingthe gene for ADP; expression of ADP from another promoter, e.g. thehuman cytomegalovirus promoter, inserted into a variety of sites in theAd genome; and insertion of the gene for ADP behind the gene for anotherAd mRNA, together with a sequence on the 5′ side of the ADP sequencethat allows for internal initiation of translation of ADP, e.g. the Adtripartite leader or a viral internal ribosome initiation sequence.

The ADP expressed by a vector according to the invention is anypolypeptide comprising a naturally-occurring full-length ADP amino acidsequence or variant thereof that confers upon a vector expressing theADP the ability to lyse a cell containing the vector such thatreplicated copies of the vector are released from the infected cell. Apreferred full-length ADP comprises the ADP amino acid sequence encodedby Ad1, Ad2, Ad5 or Ad6. These naturally-occurring ADP sequences are setforth in SEQ ID NOS:5-8, respectively. ADP variants include fragmentsand deletion mutants of naturally-occurring adenovirus death proteins,as well as full-length molecules, fragments and deletion mutantscontaining conservative amino acid substitutions, provided that suchvariants retain the ability, when expressed by a vector inside a cell,to lyse the cell.

Conservative amino acid substitutions refer to the interchangeability ofresidues having similar side chains. Conservatively substituted aminoacids can be grouped according to the chemical properties of their sidechains. For example, one grouping of amino acids includes those aminoacids having neutral and hydrophobic side chains (A, V, L, I, P, W, F,and M); another grouping is those amino acids having neutral and polarside chains (G, S, T, Y, C, N, and Q); another grouping is those aminoacids having basic side chains (K, R, and H); another grouping is thoseamino acids having acidic side chains (D and E); another grouping isthose amino acids having aliphatic side chains (G, A, V, L, and I);another grouping is those amino acids having aliphatic-hydroxyl sidechains (S and T); another grouping is those amino acids havingamine-containing side chains (N, Q, K, R, and H); another grouping isthose amino acids having aromatic side chains (F, Y, and W); and anothergrouping is those amino acids having sulfur-containing side chains (Cand M). Preferred conservative amino acid substitutions groups are: R-K;E-D, Y-F, L-M; V-I, and Q-H.

As used herein, an ADP variant can also include modifications of anaturally-occurring ADP in which one or more amino acids have beeninserted, deleted or replaced with a different amino acid or a modifiedor unusual amino acid, as well as modifications such as glycosylation orphosphorylation of one or more amino acids so long as the ADP variantcontaining the modified sequence retains cell lysing activity.

As described below, the inventors herein performed a structure-functionanalysis of ADP which defined specific domains in ADP required topromote cell death. Using this information, when combined with knownrecombinant DNA and cloning methodology, it is believed the skilledartisan can readily construct ADP variants of a naturally-occurringadenovirus death protein and test them for cell lysing activity. Apreferred ADP deletion mutant comprises an ADP amino acid sequence fromany of the deletion mutants dl716, dl715, dl714 and dl737, whose ADPsequences are set forth in SEQ ID NOS:9-12, respectively).

Where the vector is derived from a virus, it is preferred that the viruslack expression of one or more viral proteins involved in avoiding hostanti-viral defenses such as immune-mediated inflammation and/orapoptosis of infected cells. For example, adenovirus contains a cassetteof genes that prevents killing of Ad-infected cells by the immune system(Wold et al., Semin. Virol., 1998(8:515-523, 1998). The E3-14.7K proteinand the E3 RID (Receptor Internalization and Degradation) protein, whichis a complex consisting of RIDα and RIDβ, inhibit apoptosis ofAd-infected cells induced by tumor necrosis factor (TNF) and the Fasligand which are expressed on, or secreted by, activated macrophages,natural killer (NK) cells, and cytotoxic lymphocytes (CTLs) (Tollefsonet al., Nature 392:727-730, 1998). The E3-gp19K protein inhibitsCTL-killing of infected cells by blocking transport of MHC class Iantigens to the cell surface (Wold et al., supra). Thus, it is believedthat infection of tumor cells by such viral vectors will stimulateinfiltration of inflammatory cells and lymphocytes into the tumor, andwill not prevent infected tumor cells from apoptosis induced bycytolytic cells of the immune system, or against apoptosis inducingcytokines. For example, it is known that when mice are infected with Admutants lacking the E3 gp19K, RID and 14.7K proteins there is a dramaticincrease (as compared to E3-positive Ad) in infiltration of inflammatorycells and lymphocytes into the infected tissue (Sparer et al., J. Virol.70:2431-2439, 1996). A similar infiltration of tumors infected by anADP-expressing viral vector of the invention would be expected tofurther promote destruction of the tumor by adding an immune systemattack to the ADP-mediated killing activity. For example, it is believedthat the viral infection will stimulate formation of tumor-specificCTL's that can kill neoplastic cells not only in the tumor but also onesthat have metastasized. In addition, it is also expected thatvector-specific CTL's will be generated which could attackvector-infected cells if the vector spreads away from the tumor intonormal cells. Because viral vectors overexpressing ADP will spreadrapidly through the tumor, it is believed these immune mechanisms willhave little effect on spread of the vector.

Where the vector is a recombinant adenovirus, it is preferred that theadenovirus lack expression of each of the E3 gp19K, RID, and 14.7Kproteins. By “lack expression” and “lacking expression” of a protein(s),“it is meant” that the viral genome contains one or more mutations thatinactivates expression of a functional protein, i.e., one having all thefunctions of the wild-type protein. The inactivating mutation includesbut is not limited to substitution or deletion of one or morenucleotides in the encoding gene(s) that prevents expression offunctional transcripts or that results in transcripts encodingnonfunctional translation products. A particularly preferred way toinactivate expression of the Ad E3 gp19K, RID, and 14.7K proteins is bydeleting the E3 region containing the genes encoding these proteins.Preferably, one or both of the E3 genes encoding the E3 6.7K and 12.5Kproteins are also deleted because, as discussed in the Examples below,it is believed that deletion of most or all of the E3 genes other thanthe ADP gene facilitates overexpression of ADP mRNA by reducingcompetition for splicing of the major late pre-mRNAs. Preferred Advectors containing an E3 deletion that overexpress ADP are GZ1 (SEQ IDNO:3) and GZ3 (SEQ ID NO:4), whose construction and properties aredescribed in the Examples below.

The invention also provides ADP-expressing vectors whose replication isrestricted to dividing cells. Any means known to provide such areplication-restricted phenotype may be used. For example, WO 96/40238describes microbes that preferentially invade tumor cells as well asmethods for identifying and isolating bacterial promoters that areselectively activated in tumors. It is also contemplated that expressionof one or more vector proteins essential for replication can be placedunder the control of the promoter for a cellular gene whose expressionis known to be upregulated in neoplastic cells. Examples of such genesinclude but are not limited to: the breast cancer markers mammaglobin(Watson et al., Oncogene 16:817-824, 1998); BRCA1 (Norris et al., J.Biol. Chem. 270:22777-22782, 1995) and her2/neu (Scott et al., J. Biol.Chem. 269:19848-19858, 1994); and prostate specific antigen (U.S. Pat.No. 5,698,443); surfactant protein B for lung alveoli (Yan et al., J.Biol. Chem. 270:24852-24857, 1995); factor VII for liver (Greenberg etal., Proc. Natl. Acad. Sci. USA 92:12347-12351, 1995); and survivin forcancer in general (Li et al., Nature 396:580-584). Where the vector isan adenovirus, it is contemplated that such tumor-specific promoters canbe substituted for the E4 promoter. Because E4 gene products areessential for Ad replication, placing their expression under the controlof a tumor-specific promoter should restrict replication of the vectorto tumor cells in which the promoter is activated.

Another strategy for restricting replication of ADP-expressing Advectors to neoplastic cells is exemplified by the KD1 (SEQ ID NO:1), KD2(SEQ ID NO:13) and KD3 (SEQ ID NO:2) vectors, whose construction andproperties are described in the Examples below. This strategy exploits apre-existing Ad5 mutant in the E1A gene, named dl1101/1107 (Howe et al.,Proc. Natl. Acad. Sci., 87:5883-5887, 1990), also referred to herein asdl01/07, and which can only grow well in cancer cells. The role of E1Ais to drive cells from the G_(O) and G₁ phases of the cell cycle intoS-phase. This is achieved by two mechanisms, one involving pRB (andfamily members), and the other involving p300 and the related proteinCBP (DePinho, R. A., Nature 391:533-536, 1998). One domain in E1A bindsmembers of the pRB family. pRB normally exists in the cell as a complexwith the transcription factor E2F-1 and E2F family members (E2F),tethered via E2F to E2F binding sites in promoters of cells expressed inS-phase. Here, pRB acts as a transcriptional co-repressor. E1A bindingto pRB relieves this repression, and causes the release of E2F frompRB/E2F complexes. Free E2F then activates promoters of genes expressedin S-phase, e.g. thymidine kinase, ribonucleotide reductase, etc.Another domain in E1A binds the p300/CBP transcription adaptor proteincomplex. p300/CBP is a transcriptional co-activator that binds manydifferent transcription factors and accordingly is targeted topromoters. p300/CBP has intrinsic histone acetyltransferase activity.E1A binding to p300/CBP is believed to inhibit this histoneacetyltransferase activity, allowing acetylation of histones andrepression of transcription (Chakravarti et al., Cell 96:393-403, 1999;Hamamori et al., Cell 96:405-413, 1999). Conceivably, some of the genesthat are repressed as a result of E1A interacting with p300/CBP to playa role in blocking the cell cycle, although this is not known. Cancercells are cycling, so they have free E2F and presumably somep300/CBP-regulated genes are repressed. Consistent with these ideas, E1Amust bind both p300/CBP and the pRB family in order to transform primarycells to a constitutively cycling state (Howe et al., supra). The mutantdl01/07 lacks both the p300/CBP- and pRB-binding domains and, asexpected, it replicates very poorly in non-dividing “normal” cells orserum-starved cancer cells, but well in growing cancer cells. Asdescribed below, the growth of the KD1 and KD3 vectors, which containthe dl01/07 E1A mutation, is very much better in dividing cancer cellsas compared to non-dividing cells. Because the dl01/07 mutant iscompletely defective in oncogenic transformation of rat cells (Howe etla., supra), vectors according to the invention that contain this E1Amutation cannot induce cancer in humans (remote as that may be) throughan E1A-dependent mechanism.

The invention also includes vectors overexpressing ADP whose replicationis restricted to specific tissues by placing expression of one or moreproteins essential for replication under the control of a tissuespecific promoter. A number of tissue-specific promoters have beendescribed in the art such as the surfactant protein B promoter which isonly active in cells containing the TTF1 transcription factor, i.e.,type II alveolar cells (Yan et al., supra) the transcriptionalregulatory element described in U.S. Pat. No. 5,466,596 to Breitman etal., that directs gene expression specifically in cells of endotheliallineage; prostate specific antigen which is expressed in prostate cells(Rodriguez et al., supra); and human alpha-lactalbum gene which isexpressed in breast cancer cells (Anderson et al., Gene Therapy6:854-864, 1999). Many other tissue-specific or tissue-preferredenhancer/promoters have been reported (Miller and Whelan, Human GeneTherapy 8:803-815, 1997).

Replication of vectors according to the invention can also be controlledby placing one or more genes essential for vector replication under thecontrol of a promoter that is activated by an exogenous inducing agent,such as metals, hormones, antibiotics, and temperature changes. Examplesof such inducible promoters include but are not limited tometallothionein promoters, the glucocorticoid promoter, the tetracyclineresponse promoter, and heat shock protein (hsp) promoters such as thehsp 65 and 70 promoters.

The invention also provides compositions comprising a recombinant vectorthat overexpresses ADP in an amount effective for promoting death ofneoplastic cells and a method comprising administering a therapeuticallyeffective amount of the vector to a neoplastic cell in a patient. It isbelieved the compositions and methods of the present invention areuseful for killing neoplastic cells of any origin and include neoplasticcells comprising tumors as well as metastatic neoplastic cells.

It is also contemplated that ADP-expressing viral vectors can beadministered to neoplastic cells along with a replication-defectivevirus that expresses an anti-cancer gene product. For example, manyreplication-defective E1⁻ Ad vectors for use in cancer therapy are wellcharacterized. A limitation of replication-defective vectors is thatthey only synthesize the therapeutic protein in the cell they initiallyinfect, they cannot spread to other cells. Also, since the genome doesnot replicate, transcription can only occur from the input genomes, andthis could be as low as one copy per cell. In contrast, the genome ofreplication-competent Ad vectors are amplified by about 10⁴ in the cellthat was initially infected, providing more templates for transcription.More amplification is achieved as the vector spreads to other cells. Bycombining replication-defective viral vectors expressing an anti-cancergene product with replication-competent viral vectors described herein,it is expected that the result will be template amplification and rapidspread of both vectors to surrounding cells. For example, with Ad-basedvectors, the burst size for each vector should be large, ˜10⁴ PFU/cell,so the probability of co-infection of surrounding cells by both vectorswill be high. Thus, both the replication-competent andreplication-defective vectors should spread simultaneously through thetumor, providing even more effective anti-cancer therapy.

Expression of the anti-cancer gene product encoded by thereplication-defective vector can be under the control of eitherconstitutive, inducible or cell-type specific promoters. The anti-cancergene product can be any substance that promotes death of a neoplasticcell. The term “gene product” as used herein refers to any biologicalproduct or products produced as a result of the biochemical reactionsthat occur under the control of a gene. The gene product can be, forexample, an RNA molecule, a peptide, a protein, or a product producedunder the control of an enzyme or other molecule that is the initialproduct of the gene, i.e., a metabolic product. For example, a gene canfirst control the synthesis of an RNA molecule which is translated bythe action of ribosomes into a prodrug converting enzyme which convertsa nontoxic prodrug administered to a cancer patient to a cell-killingagent; the RNA molecule, enzyme, and the cell-killing agent generated bythe enzyme are all gene products as the term is used here. Examples ofanti-cancer gene products include but are not limited to cell-killingagents such as apoptosis-promoting agents and toxins; prodrug convertingenzymes; angiogenesis inhibitors; and immunoregulatory molecules andantigens capable of stimulating an immune response, humoral and/orcellular, against the neoplastic cell.

Apoptosis-promoting agents include but are not limited to thepro-apoptotic members of the BCL-2 family such as BAX, BAD, BID and BIK,as well as antisense molecules which block expression of anti-apoptoticmembers of the BCL-2 family. Examples of immunoregulatory molecules arecytokines such as tumor necrosis factor, Fas/Apo1/CD95 ligand, tumornecrosis factor related apoptosis inducing ligand, interleukins,macrophage activating factor and interferon γ. Angiogenesis inhibitorsinclude but are not limited to endostatin and angiostatin. Toxinsinclude but are not limited to tumor necrosis factor, lymphotoxin, theplant toxin ricin, which is not toxic to humans due to the lack of ricinreceptors in animal cells, and the toxic subunit of bacterial toxins.Examples of pro-drug converting enzymes and pro-drug combinations aredescribed in WO 96/40238 and include: thymidine kinase and acyclovir organcyclovir; and bacterial cytosine deaminase and 5-fluorocytosine.

The therapeutic or pharmaceutical compositions of the present inventioncan be administered by any suitable route known in the art including forexample by direct injection into a tumor or by other injection routessuch as intravenous, subcutaneous, intramuscular, transdermal,intrathecal and intracerebral. Administration can be either rapid as byinjection or over a period of time as by slow infusion or administrationof slow release formulation. For treating tissues in the central nervoussystem, administration can be by injection or infusion into thecerebrospinal fluid (CSF). When it is intended that a recombinant vectorof the invention be administered to cells in the central nervous system,administration can be with one or more agents capable of promotingpenetration of the vector across the blood-brain barrier. Preferably,vectors of the invention are administered with a carrier such asliposomes or polymers containing a targeting moiety to limit delivery ofthe vector to targeted cells. Examples of targeting moieties include butare not limited to antibodies, ligands or receptors to specific cellsurface molecules.

Compositions according to the invention can be employed in the form ofpharmaceutical preparations. Such preparations are made in a manner wellknown in the pharmaceutical art. One preferred preparation utilizes avehicle of physiological saline solution, but it is contemplated thatother pharmaceutically acceptable carriers such as physiologicalconcentrations of other non-toxic salts, five percent aqueous glucosesolution, sterile water or the like may also be used. It may also bedesirable that a suitable buffer be present in the composition. Suchsolutions can, if desired, be lyophilized and stored in a sterileampoule ready for reconstitution by the addition of sterile water forready injection. The primary solvent can be aqueous or alternativelynon-aqueous.

The carrier can also contain other pharmaceutically-acceptableexcipients for modifying or maintaining the pH, osmolarity, viscosity,clarity, color, sterility, stability, rate of dissolution, or odor ofthe formulation. Similarly, the carrier may contain still otherpharmaceutically-acceptable excipients for modifying or maintainingrelease or absorption or penetration across the blood-brain barrier.Such excipients are those substances usually and customarily employed toformulate dosages for parenteral administration in either unit dosage ormulti-dose form or for direct infusion into the cerebrospinal fluid bycontinuous or periodic infusion.

It is also contemplated that certain formulations containingADP-expressing vectors are to be administered orally. Such formulationsare preferably encapsulated and formulated with suitable carriers insolid dosage forms. Some examples of suitable carriers, excipients, anddiluents include lactose, dextrose, sucrose, sorbitol, mannitol,starches, gum acacia, calcium phosphate, alginates, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, gelatin,syrup, methyl cellulose, methyl- and propylhydroxybenzoates, talc,magnesium, stearate, water, mineral oil, and the like. The formulationscan additionally include lubricating agents, wetting agents, emulsifyingand suspending agents, preserving agents, sweetening agents or flavoringagents. The compositions may be formulated so as to provide rapid,sustained, or delayed release of the active ingredients afteradministration to the patient by employing procedures well known in theart. The formulations can also contain substances that diminishproteolytic degradation and promote absorption such as, for example,surface active agents.

The specific dose is calculated according to the approximate body weightor body surface area of the patient or the volume of body space to beoccupied. The dose will also be calculated dependent upon the particularroute of administration selected. Further refinement of the calculationsnecessary to determine the appropriate dosage for treatment is routinelymade by those of ordinary skill in the art. Such calculations can bemade without undue experimentation by one skilled in the art. Exactdosages are determined in conjunction with standard dose-responsestudies. It will be understood that the amount of the compositionactually administered will be determined by a practitioner, in the lightof the relevant circumstances including the condition or conditions tobe treated, the choice of composition to be administered, the age,weight, and response of the individual patient, the severity of thepatient's symptoms, and the chosen route of administration. Doseadministration can be repeated depending upon the pharmacokineticparameters of the dosage formulation and the route of administrationused.

The invention also contemplates passively immunizing patients who havebeen treated with a viral vector overexpressing ADP. Passiveimmunization can include administering to the patient antiserum raisedagainst the viral vector, or gamma-globulin or vector-specific purifiedpolyclonal or monoclonal antibodies isolated from the antiserum.Preferably, the patient is passively immunized after a time periodsufficient for the viral vector to replicate in and spread through thetumor.

Preferred embodiments of the invention are described in the followingexamples. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims which follow the examples.

EXAMPLE 1

This example illustrates the construction and characterization of theKD1 and KD3 anti-cancer vectors.

To construct KD1, the inventors deleted the entire E3 region of a uniqueplasmid, leaving behind only a unique PacI site for cloning. Thestarting plasmid was pCRII, purchased from Invitrogen, containing theAd5 BamHIA fragment having a deletion of all the E3 genes; the E3deletion is identical to that for KD1 and GZ3, the sequences of whichare given in SEQ ID NO:1 and SEQ ID NO:4, respectively. The ADP genefrom Ad5 was cloned into the PacI site, then built into the E3 region ofthe genome of the Ad5 E1A mutant named dl01/07. This was done byco-transfecting into human embryonic kidney 293 cells the aforementionedBamHIA fragment containing the ADP gene together with the overlappingEcoRIA restriction fragment obtained from dl01/07. Complete viralgenomes are formed within the cell by overlap recombination between theAd sequences in the BamHIA fragment in the plasmid and the EcoRIAfragment. KD3 was constructed in the same way except the E3 gene for the12.5K protein was retained in the starting plasmid. A vector named KD2,which marginally overexpress ADP, was also prepared. Plaques of eachrecombinant Ad were picked, screened, purified, expanded intoCsCl-banded stocks, sequenced, titered, and characterized. GZ1 and GZ3are Ad vectors that are identical to KD1 and KD3, respectively, exceptthat GZ1 and GZ3 have wild-type E1A sequences as found in AD5 or in theAd5 mutant dl309. GZ1 and GZ3 were constructed as described for KD1 andKD3 except that the EcoRIA fragment of AdS was used for GZ1 and GZ3.

KD1 and KD3 were characterized in cell culture by infecting the humanA549 lung carcinoma cell line with high titer (1−8×10¹⁰ plaque formingunits [PFU] per ml) virus stocks of one of these recombinant vectors, orwith one of the control viruses dl01/07, dl309, dl327, and Ad5 (wt).Fifty PFU per cell were used for each virus. The descriptions of theseviruses as well as some other viruses used in these examples arepresented in Table 1.

TABLE 1 Description of mutations in viruses: RNA REGION Virus E1 VA E3E4 dl1101/1107 dl1101: deletion From dl309 From dl309 deletion of Ad5 bp28597-28602; wild type of Ad5 bp 569-634 deletion of Ad5deletion-substitution Ad5 bp 3005-30750, insert 642 dl1107: deletion bp10594-10595 bp DNA of unknown origin of Ad5 bp 890-928 KD1 dl1101:deletion From dl309 deletion of Ad5 bp 27858-27860, TAA inserted; wildtype of Ad5 bp 569-634 deletion of Ad5 deletion of Ad5 bp 27982-28134;deletion of Ad5 bp dl1107: deletion bp 10594-10595 28395-29397, insertCCTTAATTAAA (SEQ ID NO:21); of Ad5 bp 890-928 deletion of Ad5 bp29783-30883, insert TTAATTAAGG (SEQ ID NO:22) KD2 dl1101: deletion Fromdl309 dl309 background, gp19K mutated deletion of Ad5 wild type of Ad5bp 569-634 deletion of Ad5 bp 28597-28602; deletion-substitution Ad5 bp3005- dl1107: deletion bp 10594-10595 30750, insert 642 bp DNA ofunknown origin; of Ad5 bp 890-928 deletion of Ad5 bp 28788-28789, insertTTAATTAA (SEQ ID NO:23) KD3 dl1101: deletion From dl309 deletion of Ad5bp 28598-29397; deletion of Ad5 bp wild type of Ad5 bp 569-634 deletionof Ad5 29783-30469 dl1107: deletion bp 10594-10595 of Ad5 bp 890-928 GZ1wt wild type deletion of Ad5 bp 27858-27860, TAA inserted; wild typedeletion of Ad5 bp 27982-28134; deletion of Ad5 bp 28395-29397, insertCCTTAATTAAA (SEQ ID NO:24); deletion of Ad5 bp 29783-30883, insertTTAATTAAGG (SEQ ID NO:25) GZ3 wild type wild type deletion of AD5 bp28598-29397; deletion of Ad5 bp wild type 29783-30469 dl1101/1107-dl1101: deletion From dl309 From dl309 deletion of Ad5 bp 28597-28602;E4 promoter SPB of Ad5 bp 569-634 deletion of Ad5 deletion-substitutionAd5 bp 3005-30750, insert 642 deletion- dl1107: deletion bp 10594-10595bp DNA of unknown origin substitution: of Ad5 bp 890-928 deletion of Ad5bp 35623-35775, insert SP-B 500 promoter flanked by Bst1 1071 sitesKD1-SPB dl1101: deletion From dl309 deletion of Ad5 bp 27848-27860, TAAinserted; E4 promoter of Ad5 bp 569-634 deletion of Ad5 deletion of Ad5bp 27982-28134; deletion of Ad5 bp deletion- dl1107: deletion bp10594-10595 28395-29397, insert CCTTAATTAAA (SEQ ID NO:26);substitution: of Ad5 bp 890-928 deletion of Ad5 bp 29783-30883, insertTTAATTAAGG deletion of Ad5 bp (SEQ ID NO:27) 35623-35775, insert SP-B500 promoter flanked by Bst1 1071 sites KD3-SPB dl1101: deletion Fromdl309 deletion of Ad5 bp 28598-29397; deletion of Ad5 bp E4 promoter ofAd5 bp 569-634 deletion of Ad5 29783-30469 deletion- dl1107: deletion bp10594-10595 substitution: of Ad5 bp 890-928 deletion of Ad5 bp35623-35775, insert SP-B 500 promoter flanked by Bst1 1071 sites

Using a polymerase chain reaction (PCR)-based protocol, an in-frame stopcodon was introduced into the gene for the E3-gp19K protein in the E3region of the Ad5 mutant dl309 (Jones and Shenk, Cell 17:683-689, 1979).The mutagenesis was conducted using a SunI-Bst1107I fragment,nucleotides 28,390 to 29,012 in the Ad5 genome, which was thensubstituted for the equivalent fragment in dl309. dl01/07 is the parentfor KD1 and KD3. In turn, the Ad5 mutant named dl309 is the parent ofdl01/07, i.e. dl309 is identical to dl01/07 except that dl309 does nothave the E1A mutation. Both dl01/07 and dl309 have deletions of thegenes for the E3 RIDα, RIDβ and 14.7K proteins but retain the gene forADP. The Ad5 mutant dl327 has wild-type E1A, it lacks the gene for ADP,and its lacks all other E3 genes except the one for the 12.5K protein.

At 24 and 36 hours post-infection (h p.i.), proteins were extracted fromthe A549 cells and analyzed for ADP by immunoblot using a rabbitantiserum against ADP (Tollefson et al., J. Virol. 66:3633-3642, 1992).The results are shown in FIG. 2. Much more ADP was detected at 24 and 36h p.i. in KD1- and KD3-infected cells than in cells infected withdl01/07. Also, much more ADP was synthesized by GZ1 and GZ3 than dl309or the other viruses. Most importantly, KD1, KD3, GZ1, and GZ3 expressedmuch more ADP at 24 h p.i. than did dl01/07 or dl309 (FIG. 2). Thisresult is consistent with an observation discussed below that the cellsinfected with KD1, KD3, GZ1, or GZ3 lyse faster, and that these virusesspread from cell to cell faster than dl01/07 or dl309. It is noteworthythat KD1, KD3, GZ1, and GZ3 express much more ADP at 24 and 36 h p.i.than the Ad5 mutant dl1520 (FIG. 2); dl1520 is the original name givento ONYX-015 (Heise et al., Nature Medicine 3:639-645, 1997). Asexpected, no ADP was detected in cells infected with pm734.1 (FIG. 2), amutant that lacks amino acids 1 to 48 in ADP (Tollefson et al., J.Virol. 70:2296-2306, 1996). Expression of the E1A proteins by dl01/07,KD1, KD2, and KD3 was slightly less than by Ad5, dl309, or dl327, and asexpected from the dl01/07 deletion, the proteins were smaller (FIG. 3A).dl327 is isogenic with dl324 (Thimmappaya et al., 1982 Cell 31:543-51,1983), and it lacks the gene for ADP and all other E3 proteins exceptthe 12.5K protein.

The amount of ADP detected in the KD1 and KD3 infected cells issignificantly higher than the amount detected in the dl309 infectedcells (FIG. 2). If one takes into consideration the fact that theviruses with the E1A mutation replicate somewhat slower, as evidenced inby the delayed appearance of the late proteins (FIG. 3B), it is clearthat KD1 and KD3 express much more ADP per viral genome present in thecell than dl309. This finding is supported by the fact that when A549cells are coinfected with a virus containing the E1A mutation and dl327,which lacks ADP but has wild-type E1A, the replication rates of the E1Amutant viruses speed up, as indicated by earlier appearance of lateproteins (compare FIGS. 3B and 3D). Thus, dl327 complements the E1Amutation. In conclusion, these experiments demonstrate that ADP isdramatically overexpressed by KD1, KD3, GZ1, and GZ3. ADP is marginallyoverexpressed by KD2 (not shown).

EXAMPLE 2

This example illustrates that KD1 and KD3 lyse cells more rapidly andspread from cell-to cell faster than other adenoviruses.

The ability of KD1 and KD3 to lyse cells was examined by a trypan blueexclusion cell viability assay which was performed essentially asdescribed by Tollefson et al., J. Virol. 70:2296-2306, 1996. In brief,A549 cells were mock-infected or infected with 20 PFU/cell of KD1, KD3,dl01/07, dl327 or dl309. At various days p.i., the number of viablecells was determined using a hemacytometer (600 to 1000 cells werecounted per time point) and the results are shown in FIG. 4.

Only 25% of the KD1-infected cells and 9% of the KD3-infected cells werealive at 5 days p.i. as compared to 44% of cells infected with dl01/07,which has the same E1A mutation as KD1 and KD3. The KD1 and KD3 vectorsalso lysed cells faster than dl309, which has a wild-type E1A region.When infected with dl327 (ADP⁻, E1A⁺), 94% of the cells were alive after5 days. When cell lysis was estimated by release of lactatedehydrogenase, KD1 and KD3 once again lysed cells faster than dl01/07and dl309, and dl327 caused little cell lysis (data not shown). Thus,ADP is required for efficient cell lysis, and over-expression of ADPincreases the rate of cell lysis.

As another means to measure cell lysis and to examine virus replicationin cancer cells, separate groups of A549 cells were infected with 20PFU/cell of KD1, KD3, dl01/07, or dl309 and the amount of intracellularand extracellular virus was determined by plaque assay on A549 cells. At2 days p.i., the total amount of virus formed in each group was similar,2−4×10⁸ PFU/ml, indicating that replication of all the viruses issimilar. However, when the ratio of extracellular to intracellular viruswas calculated, the value for KD1 and KD3 was 2-3 logs higher than forAd5, dl309, or dl01/07 (data not shown). Thus, virus is released muchmore rapidly from cells infected with KD1 and KD3, which overexpressADP, than with viruses expressing wild-type amounts of ADP.

The ability of KD1 and KD3 to spread from cell-to-cell was measured in a“cell spreading” assay. In this assay monolayers of A549 cells in a 48well culture dish were mock-infected or infected with 10⁻³, 10⁻², 10⁻¹,10⁰, or 10 PFU/cell of dl327, dl309, Ad5, dl01/07, KD1 or KD3. At lowPFU/cell, the viruses must go through two or three rounds of replicationin order to infect every cell in the monolayer. At 1.0 and 10 PFU/cell,the monolayer should be destroyed by the virus that initially infectedthe cells. To assess the amount of spread in the monolayers by 7 daysp.i., crystal violet, which stains live cells but not dead cells, wasadded to the monolayers. The results are shown in FIG. 5.

Remarkably, at 7 days p.i., the monolayer was virtually eliminated byKD1 and KD3 at 10⁻³ PFU/cell, whereas 1.0 PFU/cell was required withdl01/07, dl309 and Ad5. This result attests to the potency of ADP inmediating cell lysis and virus spread in A549 cells. KD1 and KD3 arealso more effective that dl01/07 in killing other types of human cancercell lines (most purchased from the American Type Culture Collection[ATCC]) as determined in this cell spreading assay. KD1 and/or KD3killed HeLa (cervical carcinoma), DU145 (prostate), and pC3 (prostate)cells at 10⁻² PFU/cell, ME-180 (cervix) and Hep3B (liver) at 10⁻¹PFU/cell, and U118 (glioblastoma) and U373 (glioblastoma) at 10PFU/cell. From 10- to 100-fold more dl01/07 was required to kill thesecells (data not shown). These results indicate that KD1 and KD3 may beeffective against many types of cancer.

An important aspect of the finding that ADP overexpressing vectors lysecells at very low multiplicities of infection is that the multiplicityof infection in human tumors is likely to be low at sites distal to thesight of vector injection or distal to blood vessels that carry thevector to the tumor. Thus, ADP overexpressing vectors have an advantageover vectors that express less ADP or no ADP at all.

EXAMPLE 3

This example illustrates that KD1 and KD3 replicate poorly innon-growing non-cancerous cells. The replication phenotype of KD1 andKD3 was evaluated using “normal” HEL-299 human fibroblast cells, eithergrowing in 10% serum or rendered quiescent using 0.1% serum. All Adsshould replicate well in growing cells, but viruses with the dl01/07 E1Amutation should do poorly in quiescent cells because E1A is required todrive them out of G₀. dl309, which has wild-type E1A, should replicatewell in both growing and growth-arrested cells.

Cells were infected with 100 PFU/cell of KD1, KD3, dl01/07, or dl309. Atdifferent days p.i., virus was extracted and titered. In 10% serum, KD1,KD3, and dl01/07 replicated well, reaching titers of 10⁻⁶-10⁷ PFU/ml,only slightly less than dl309 (FIG. 6). However, in quiescent cells,replication of KD1, KD3, and dl01/07 was 1.5-2 logs lower than ingrowing cells, ranging from 10⁴ to 2×10⁵ PFU/ml. The titer of dl309reached 10⁷ PFU/ml, nearly the level achieved in growing cells. At 10days p.i., quiescent HEL-299 cell monolayers infected with 100 PFU/cellof KD1, KD3, or dl01/07 were intact, whereas those infected with dl309or dl327, which have wild-type E1A, showed strong typical Ad cytopathiceffect indicative of cell death (data not shown). Thus, replication ofKD1 and KD3 is severely restricted to growing cell lines.

The restriction associated with the dl01/07 E1A mutation was also testedin primary human cells (purchased from Clonetics) growing as monolayers.Bronchial epithelial cells (FIG. 7) and small airway epithelial cellswere not killed by 10 PFU/cell of KD1, KD3, or dl01/07 at 5 days p.i.,whereas they were killed by 10 PFU/cell of dl309 or dl327 (data notshown). Lung endothelial cells also were not killed after 10 days byKD1, KD3, or dl01/07 at 10 PFU/cell, but they were killed by 1 PFU/cellof dl309. These monolayers were subconfluent when initially infected,then grew to confluency. The exciting result here is that although theseprimary cells were growing, they did not support replication in thistime frame and were not killed by KD1 or KD3. Thus, it is believed thesevectors will be restricted to cancerous cells, and will have little tono effect on cells such as basal cells that are normally dividing in thebody. In addition, it is unlikely that KD1 and KD3 will affect dividingleukocytes because such cells are poorly infected by Ad.

In summary, the above experiments demonstrate that KD1 and KD3 lysecancer cells, spread from cell-to-cell rapidly, and replicate poorly inquiescent and non-cancerous cells. These properties should make themuseful in anti-cancer therapy.

EXAMPLE 4

This example illustrates that KD1 and KD3 inhibit the growth of humantumors in an animal model.

We could not evaluate mouse or rat tumors in normal mice or rats becausethey are totally non-permissive. Human cancer cell lines growing in nudemice have been used by Onyx Pharmaceuticals (Richmond, Calif.) toevaluate the efficacy of ONYX-015, an Ad vector lacking expression ofthe E1B 55 kDa protein (Heise et al., Nature Med. 3:639-645, 1997). Wehave found that A549 cells, which were used in many of our cell culturestudies, form excellent rapidly growing solid tumors when injectedsubcutaneously into nude mice. The average tumor reaches ca. 500 μl infour weeks, and is encapsulated, vascularized, and attached to the mouseskin (usually) or muscle.

Nude mice were inoculated into each hind flank with 2×10⁷ A549 cells.After 1 week tumors had formed, ranging in size from about 20 μl to 50μl. Individual tumors were injected three days later, and at subsequentweeks for 4 weeks (total of 5 injections), with 50 μl of buffer or 50 μlof buffer containing 5×10⁷ PFU of dl309, dl01/07, KD1, KD3, or pm734.1,with a total virus dose per tumor of 3×10⁸ PFU. The mutant pm734.1 lacksADP activity due to two nonsense mutations in the gene for ADP, but allother Ad proteins are expected to be synthesized at wild-type levels(Tollefson et al., J. Virol. 70:2296-2306, 1996). The efficacy of eachvirus (or buffer) was tested on six tumors. At weekly intervals, thelength (L) and width (W) of tumors were measured using a Mitutoyodigital caliper. Tumor volumes were calculated by multiplying L×W×W/2.This value was divided by the tumor volume at the time of the initialvirus injection, the fold-increase in tumor growth was calculated, andthe average for the six tumors was graphed.

As shown in FIG. 8A, tumors that received buffer continued to grow,increasing about 14-fold by 5 weeks. In contrast, tumors injected withdl309, which expresses normal amounts of ADP and lacks the E3 RID and14.7K and proteins, only grew about 2.5-fold by 5 weeks. With pm734.1,which lacks ADP, the tumors grew as well as those that received buffer.Thus, dl309 markedly decreases the rate of tumor growth, and ADP isrequired for this decrease. Tumors inoculated with dl01/07 grew about8-fold over 5 weeks. Since dl01/07 is identical to dl309 except for theE1A mutation, this result indicates that the E1A mutation significantlyreduces the ability of Ad to prevent growth of the tumors. This effectis probably due to a reduction in virus replication in the tumorsresulting in lower ADP expression, but it could also reflect otherproperties of E1A in the tumor cells, e.g. the inability of the mutantE1A proteins to induce apoptosis. Most importantly, tumors inoculatedwith KD1 or KD3 only grew about 2.5-fold. Thus, the overexpression ofADP by KD1 and KD3 allows KD1 and KD3 to reduce tumor growth to a ratemarkedly slower than dl01/07 (their parental control virus), and even toa rate similar to that of dl309.

The finding that KD1 and KD3 are as effective as wild-type Ad (i.e.dl309) in reducing the rate of A549 tumor growth is highly significantin the context of cancer treatment, inasmuch as KD1 and KD3 arerestricted to cancer cells whereas wild-type Ad does not have such arestriction.

The tumors in FIG. 8A received five injections of vectors, but only onedose of vector, in this case 5×10⁸ of each of KD3 or GZ3, is sufficientto significantly reduce the rate of A549 tumor growth (FIG. 8B).

We have also found that KD1 and KD3 reduce the rate of growth in nudemice of a human liver cancer cell line, Hep3B cells. These cells formrapidly growing tumors that are highly vascularized. Nude mice wereinoculated into each hind flank with 1×10⁷ of Hep3B cells. After tumorsreached about 100 μl, they were injected twice per week for 3 weeks with50 μl of buffer or 5×10⁷ PFU of KD1, KD3, or dl309. There were typically8-10 tumors per test virus. The tumor sizes were measured and the foldincrease in size at 0 to 3.5 following the initial virus injection wasgraphed as described above for the A549 tumors. Tumors that receivedbuffer alone grew 9-fold over 3 weeks and were projected to grow about12-fold over 3.5 weeks (after 3 weeks the mice had to be sacrificedbecause the tumors were becoming too large) (FIG. 9). Tumors thatreceived KD1 or KD3 grew about 4-fold, establishing that KD1 and KD3reduce the growth of Hep3B tumors in nude mice. Tumors that wereinjected with dl309 grew 2-fold (FIG. 9). The finding that KD1 and KD3were somewhat less effective than dl309 is probably due to the fact thatthey do not grow as well as dl309 in Hep3B cells, as indicated by a cellspread assay in culture (data not shown). In any case, the importantpoints are that KD1 and KD3 are effective against the Hep3B tumors, andthat they contain the E1A mutation that limits their replication tocancer cells.

These results point to the potency of ADP as an anti-tumor agent whenexpressed in an Ad vector. It is highly probable that KD1 and KD3 willprovide significant clinical benefit when used to infect tumors growingin humans.

EXAMPLE 5

This example illustrates the use of replication-defective Ad vectors incombination with KD1 or KD3.

It is well established that replication-competent (RC) virusescomplement replication-defective (RD) mutants. That is, when the samecell is infected, the competent virus will supply the protein(s) thatcannot be made from the mutant genome, and both viruses will grow. Totest the ability of KD1 and KD3 to complement RD viruses, two RD vectorsexpressing β-galactosidase were constructed. The first, named Ad-β-gal,has a cDNA encoding β-gal under the control of the Rous Sarcoma Viruspromoter substituted for the deleted E1 region. Ad-β-gal also has the E3region deleted, including the gene for ADP. The second, namedAd-β-gal/FasL is identical to Ad-β-gal, except that it also expressesmurine FasL from the human cytomegalovirus promoter/enhancer. Thesevectors were constructed by overlap recombination in human 293 cellsthat constitutively express the Ad E1A and E1B genes and complementreplication of the E1-minus vectors.

These RD vectors should infect and express β-gal in A549 cells, butshould not replicate because the E1A proteins are lacking. However, thevectors should replicate when cells are co-infected with RC Ads. Toprove this, A549 cells were infected with 10 PFU/cell of Ad-β-gal alone,or with 10 PFU/cell of Ad-β-gal plus 10 PFU/cell of KD1, KD3, dl01/07,dl309, or dl327. At 2 days p.i., virus was extracted and Ad-β-gal titersdetermined by β-gal expression in A549 cells. The yields are shown inTable 2 below.

TABLE 2 Yield Virus (blue plaques per ml) Ad-β-gal 1 × 10² Ad-β-gal +KD1 2 × 10⁵ Ad-β-gal + KD3 3 × 10⁵ Ad-β-gal + dl01/07 4 × 10⁴ Ad-β-gal +dl309 3 × 10⁵ Ad-β-gal + dl327 3.0 × 10⁵  The data in Table 2 indicate that the complementing viruses increasedthe yield of Ad-β-gal by about 10³.

A key feature of KD1 and KD3 is that they spread from cell-to-cellfaster than other Ads. Accordingly, they should complement the spread ofAd-β-gal. To test this, an infectious center assay was conducted. A549cells were infected with Ad-β-gal plus KD1, KD3, or dl01/07. After 2 h,cells were collected, diluted, and seeded onto monolayers of fresh A549cells. After 4 days, the cells were stained with X-gal and the resultsare shown in FIG. 10.

With Ad-β-gal alone, only the originally infected cell (before seeding)should be stained, and the vector should not spread to other cells onthe seeded monolayer. This was indeed the case. In monolayers seededwith A549 cells infected with Ad-β-gal alone (dish shown in the top leftof FIG. 10A) contained a number of individual blue cells (not visible inthe print); examples are shown in the enlarged view FIG. 10B. However,when the monolayers were seeded with A549 cells coinfected with Ad-β-galand KD1 or KD3, there were numerous “comets” of blue cells (FIG. 10A).Each comet represents Ad-β-gal which has spread from oneinitially-infected cell. Most of the cells within a comet were stainedwith X-gal (FIG. 10C). Comets were also observed with dl01/07, but notto the extent of KD1 and KD3 (FIG. 10A). With dl327 (ADP⁻), there waslittle spread from the originally infected cell (data not shown). Insummary, KD1 and KD3 not only complement the replication of Ad-β-gal,they also enhance its rapid spread.

It is expected that KD1 and KD3 will also complement and enhance thespread of RD vectors expressing anti-cancer therapeutic gene products,and this expectation can be readily verified using the Ad-β-gal/FasL inreplication and infectious center assays as described above.

KD1 and KD3 not only complement the replication of RD vectors in cellculture, they also do so in Hep3B tumors growing in the hind flanks ofnude mice. The RD vector used was AdLuc, an Ad that lacks the E1 and E3regions, and has inserted into the E1 region an expression cassettewhere the firefly luciferase gene is expressed from the Rous SarcomaVirus promoter (Harrod et al., Human Gene Therapy 9:1885-1898, 1998).The Hep3B tumors were injected with 1×10⁷ PFU of AdLuc plus buffer, or1×10⁷ PFU of AdLuc plus 5×10⁷ PFU of KD1, KD3, dl01/07, or dl309. After2 weeks, mice were sacrificed and tumors excised. Proteins wereextracted from the tumors and luciferase activity determined using aluminometer. The luciferase counts per tumor were 6,800 for AdLuc plusbuffer, 113,500 for KD1, and 146,900 for KD3 (FIG. 11). Thus, KD3 andKD1 respectively caused a 22-fold and 17-fold increase in luciferaseactivity. This increase could be due to elevated synthesis of luciferasein cells that were initially coinfected the AdLuc and KD1 or KD3, and itcould also be due to spread of AdLuc from cell to cell in the tumor asmediated by KD1 or KD3.

In summary, infecting a tumor with a replication-competentADP-overexpressing vector according to the invention together with a RDvector expressing an anti-cancer gene product should greatly increasethe amount of anti-cancer protein synthesized in the tumor therebyincreasing the ability of the replication-defective vector to promotedestruction of the tumor.

EXAMPLE 6

This example illustrates the construction and characterization of arecombinant Ad vector according to the invention which isreplication-restricted to cancerous type II alveolar cells.

As demonstrated above, the dl01/07 mutation in KD1 and KD3 limits growthof these vectors to cancer cells. To further restrict their replicationphenotype, the E4 promoter in each virus was deleted and replaced by thesurfactant protein B (SPB) promoter to produce vectors named KD1-SPB(SEQ ID NO:14), KD3-SPB (SEQ ID NO:15), and dl01/07-SPB (SEQ ID NO:16).The SPB promoter is only active in cells containing the TTF1transcription factor, which has thus far been found primarily in type IIalveolar cells of the human lung (Lazzaro et al., Development113:1093-1104, 1991). Thus, KD1-SPB, KD3-SPB, and dl01/07-SPB should beseverely restricted to cancerous type II alveolar cells of the humanlung. Many lung cancers are of this type.

The KD1-SPB and KD3-SPB vectors were prepared as follows. The E4promoter is located at the right end of the Ad genome (FIG. 1). Using apCRII-based plasmid (Invitrogen) containing the Ad5 DNA sequences fromthe BamHI site (59 map units) to the right hand end of the genome, andusing and a PCR-based protocol, nearly all the transcription factorbinding sites were deleted from the E4 promoter Ad5 base pairs 35,623 to35,775 and replaced with a 500 base pair fragment containing the SPBpromoter (Yan et al., J. Biol. Chem. 270:24852-24857, 1995). The finalplasmids contain the E4-SPB substitution in the E4 region and thedl01/07, KD1, or KD3 versions of the E3 region, respectively, for theviruses dl01/07-SPF, KD1-SPB, and KD3-SPB. These plasmids wereco-transfected into 293 cells with a fragment containing the leftportion of the genome of dl01/07, and plaques were allowed to develop.Plaques were screened for the expected features, purified, then expandedinto a stock.

The A549-TTF1 cell line was developed in order to test the predictionthat replication of dl01/07-SPB, KD1-SPB, and KD3-SPB would berestricted to cancerous cells expressing the TTF1 transcription factor.These cells were co-transfected with two plasmids, one in which TTF1 isexpressed from the CMV promoter, and the other coding for resistance toneomycin Resistant clones were isolated and shown to express TTF1activity as determined by transient transfection with a plasmidexpressing chloramphenicol acetyltransferase from the TTF1-requiringsurfactant protein C promoter.

KD1-SPB and KD1 were subjected to a standard plaque development assay onA549-TTF1 cells and parental A549 cells. The results are shown in FIG.12. With KD1-SPB on A549 cells, plaques were not visible after 8 days,only about 4% of the final number of plaques were seen after 10 days,and about 50% of final plaques were seen after 12 days. With KD1-SPB onA549-TTF1 cells, plaques were visible after 6 days, and about 60% ofplaques were seen after 10 days. Thus, as expected, KD1-SPB grewsignificantly faster on the cells containing TTF1. KD1 formed plaquesmore quickly than KD1-SPB on both A549 and A549-TTF1 cells, indicatingthat the E4 promoter-SPB substitution is not as effective the wild-typeE4 promoter in inducing Ad replication. However, this difference betweenKD1-SPB and KD1 on A549-TTF1 cells is tolerable, with KD1-SPB delayedonly about 1 day. Curiously, the final titer obtained for all virusstocks by day 16 was similar, indicating that A549 cells may contain avery small amount of endogenous TTF1 activity. It is predicted thatKD3-SPB and dl01/07-SPB will behave similarly to KD1-SPB when grown inA549-TTF1 cells and A549 cells.

The restriction of KD1-SPB to cells containing TTF1 was further examinedin a cell spread assay using H441 cells, a TTF1-expressing humanpulmonary adenocarcinoma cell line (Yan et al., supra), and Hep3B cells,a liver cancer cell line not expected to express TTF1. Culture dishwells containing H441 or Hep3B cells were infected with KD1-SPB or KD1at multiplicities ranging from 10 to 10⁻⁴ PFU/cell. The H441 and Hep3Bcells were stained with crystal violet at 5 days and 8 days p.i.,respectively. KD1-SPB and KD1 grew and spread equally well on H441cells, causing destruction of the monolayer at 10⁻¹ PFU per cell (FIG.13). (Some of the H441 monolayer has peeled off in the well with KD1-SPBat 10⁻² PFU per cell, and in the wells with KD1 and KD1-SPB at 10⁻⁴ PFUper cell; this occasionally occurs in cell spread assays, and it doesnot reflect virus infection). With Hep3B cells, KD1 grew and spread verymuch better than KD1-SPB, with 10⁻² PFU per cell of KD1 causing moredestruction of the monolayer as 1.0 PFU per cell of KD1-SPB (FIG. 13).

In summary, this example demonstrates that a replication-competent Ad,which replicates well on cells expressing the appropriate transcriptionfactor, can be constructed with a tissue-specific promoter substitutedin place of the E4 promoter. This methodology should be applicable tomany other tissue specific and cell type specific promoters. Onepossibility would be a liver-specific promoter. Another possibilitywould be to use the E2F promoter, or another promoter with E2F sites,inasmuch as that promoter would be active only in cells such as cancercells that have free E2F. A third possibility would be to use aregulatable promoter, e.g. the synthetic tetracycline response promoter(Massie et al., J. Virol. 72:2289-2296, 1998), where the activity of thepromoter is controlled by the level of tetracycline or a tetracyclinanalog in the patient.

EXAMPLE 7

This example illustrates the construction and characterization ofvectors which overexpress ADP and are not replication restricted.

As demonstrated above, the dl01/07 E1A mutation in KD1 and KD3 isattenuating, inhibiting growth in non-dividing and even in dividingprimary human epithelial and endothelial cells. Ads with this mutationare able to replicate well in dividing cancer cells. However,replication of such E1A mutants is not as efficient as, e.g. dl309 whichhas a wild-type E1A gene. For instance, the rate of replication ofdl01/07, as determined by the rate at which plaques develop, is reducedsuch that dl01/07 plaques appear one day later than those of dl309 (datanot shown). This delay is due in part to a delay in expression of Adlate genes (see FIG. 3). The idea that the dl01/07 mutation retards therate of replication in A549 cells is further supported by the data inFIG. 8A, where dl01/07 did not prevent tumor growth nearly as well asdl309. Despite this negative effect of the dl01/07 E1A mutation, thereare theoretical and practical aspects of having this mutation in the KD1and KD3 vectors, as has been discussed. Nevertheless, one can easilyimagine scenarios (e.g. patients with terminal cancer) where the abilityof an Ad vector to destroy the tumor supercedes the requirement that thevector be totally restricted to tumor cells. In such cases, it would beadvantageous to have vectors similar to KD1 and KD3, but with thewild-type E1A gene. The rates at which such vectors express their genes,lyse cells, and spread from cell to cell should be higher than those ofKD1 and KD3. Such vectors might cause some damage to non-cancerous cellsand tissue, but this is also true for other modes of anti-cancertreatment such as surgery, chemotherapy, and radiation therapy.

In light of these considerations, vectors named GZ1 and GZ3 have beenconstructed that are identical to KD1 and KD3, respectively, except theyhave a wild-type E1A region. These vectors were constructed by overlaprecombination in A549 cells. The left hand fragment contained thewild-type E1A region of Ad5, and the right end fragment contained the E3modifications of KD1 or KD3. Plaques were picked, analyzed for theexpected genotype, plaque-purified, and expanded into CsCl-bandedstocks. The titers of these stocks on A549 cells were 2.9×10¹⁰ PFU/mlfor GZ1 and 1.6×10¹¹ PFU/ml for GZ3. Thus, these vectors can be growninto high titer stocks comparable to wild-type Ad. The GZ1 and GZ3plaques are larger and appear much sooner than the plaques for dl309.Large rapidly-appearing plaques reflect the ability of Ad to lyse cellsand spread from cell-to-cell (Tollefson et al., J. Virol. 70:2296-2306,1996; Tollefson et al., Virology 220:152-162, 1996), and this property,as discussed, is due to the function of ADP.

The rate of plaque appearance can be quantitated in a plaque developmentassay (Tollefson et al., supra). Here, a typical plaque assay isperformed, and the plaques observed on subsequent days of the assay arecalculated as a percentage of the number of plaques observed at the endof the plaque assay. As shown in FIG. 14, after 4 days of plaque assayon A549 cells, GZ1 and GZ3 had 48% and 34%, respectively, of the finalnumber of plaques, whereas dl309 had only 1%. It is very unusual in Adplaque assays in A549 cells for plaques to appear after only 4 days.These large plaques reflect the overexpression of ADP. These GZ1 and GZ3plaques appear sooner than those of KD1 and KD3 (data not shown), nodoubt because GZ1 and GZ3 replicate faster because they have a wild-typeE1A region.

GZ1 and GZ3 lyse cells and spread from cell to cell much moreeffectively than dl309. At 6 days p.i. of A549 cells, approximately asmuch monolayer destruction was observed with GZ1 and GZ3 at 10⁻³ PFU percell as was observed with dl309 at 10⁻¹ PFU per cell (FIG. 15, toppanel). This result further underscores the conclusion thatoverexpression of ADP promotes cell lysis and virus spread.

In theory, GZ1 and GZ3 should be able to replicate not only in tumorcells but also in normal cells. Although they can replicate in normalcells, it is quite possible that GZ1 and GZ3 may be useful asanti-cancer vectors. First, GZ1 and GZ3 could be injected directly intothe tumor. Many tumors are self-contained (encapsulated) except for theblood supply. The physical barriers of the tumor could minimizedissemination of the virus to other tissues. Second, Ads are in generalquite benign. Most infections of Ad5 are in infants and result in mildor asymptomatic disease, and are held in check by strong humoral andcellular immunity. Anti-Ad immunity appears to be life-long. GZ1 and GZ3could be used only in patients who have an intact immune system, andperhaps also with pre-existing anti-Ad immunity. Further, patients couldbe passively immunized against Ad, using gamma-globulin or even specificpurified anti-Ad neutralizing antibodies. Third, considering that Ad5 isa respiratory virus which most efficiently infects lung epithelial cellsdisplaying the specific Ad5 receptor (named CAR) as well as specificintegrins (e.g. a_(v) b5), replication-competent vectors derived fromAd5 may not spread efficiently in many non-cancer tissues of the body.In addition, it is believed that versions of GZ1 and GZ3 can beconstructed that have the E4 promoter substituted with a tumor-specific,tissue-specific, cell-specific, or synthetic promoter. Such vectorswould have the positive features associated with wild-type E1A and ADP,and yet be replication-restricted to tumor tissue and/or to particularcell types.

EXAMPLE 8

This example illustrates that the combination of KD1, KD3, GZ1, or GZ3with radiation is more effective in destroying A549 cells, growing inculture or growing as tumors in nude mice, than the vectors alone orradiation alone.

This was shown in a cell spread assay. A549 cells growing in three 48well culture dishes were mock-infected or infected with differentviruses at multiplicites of infection ranging from 10 to 10⁻⁴ pFU percell as indicated in FIG. 15. One dish was not radiated. A second dishreceived 600 centrigreys (cGy) of radiation at 24 h p.i., and a thirddish received 2000 cGy of radiation at the same time. All dishes werestained with crystal violet at 6 days p.i. With the cells that were notradiated (top panel in FIG. 15), KD1 and KD3 caused monolayerdestruction at lower multiplicities of infection than their parentalcontrol, dl01/07. This was also true for GZ1 and GZ3 as compared totheir parental control dl309. (The paucity of cells in the cellsinfected with GZ1 or GZ3 at 10⁻⁴ PFU per cell is an experimentalartifact, and is not caused by infection by GZ1 or GZ3). These KD1, KD3,GZ1 and GZ3 results are consistent with earlier results showing thatoverexpression of ADP leads to increased cell lysis and virus spread.

With the dish that was infected then radiated with 600 cGy there wasmarkedly increased cell killing and virus spread as compared to thenon-radiated cells (compare the bottom panel of FIG. 15 with the toppanel). For example, with KD1, KD3, GZ1, and GZ3 there was about thesame amount of cell destruction in the radiated wells at 10⁻⁴ PFU percell as in the non-radiated wells at 10⁻² PFU per cell. Similar resultswere seen with the dish that received 2000 cGy of radiation (data notshown), and also with dishes that received 600 or 2000 cGy of radiation24 h prior to infection (data not shown).

The amount of cell destruction was quantitated by extracting the crystalviolet from the cells with 33% acetic acid, then measuring theabsorbance at 490 nm (data not shown). The absorbance with non-radiatedmock-infected cells was set at 100% cell viability. With mock-infectedcells that received 600 cGy there was a 15% loss in viability (i.e. 15%less crystal violet was extracted). With KD1 at 10⁻³ PFU per cell, thenon-radiated cells were 80% viable whereas the cells receiving 600 cGyof radiation were only about 30% viable. Similar differences inviability between radiated and non-radiated cells were seen with KD3,GZ1, and GZ3. These results argue that the combination of radiation plusvector has a syngergistic effect on cell lysis and vector spread, ratherthan an additive effect. If the effect were only additive, then with theKD1 samples at 10⁻³ PFU per cell, the cell viability should have been65% (15% reduction in viability due to radiation alone, 20% reductiondue to KD1 alone). In fact, the cell viability was 30% rather than 65%.

As mentioned, approximately as much cell lysis and virus spread wereobserved with 600 cGy as with 2000 cGy. To determine the optimal dose ofradiation to synergize with the vectors, an experiment similar to theone described above was conducted with mock-, dl01/07-, KD1-, KD3-,dl309, GZ1-, or GZ3-infected A549 cells. The 48 well plates received 0,150, 300, or 600 cGy of radiation at 24 h p.i. Cells were stained withcrystal violet. The results with cells receiving 0 versus 600 cGy ofradiation were similar to those in FIG. 15. The crystal violet wasextracted from the cells infected with 10⁻³ PFU per cell of thedifference viruses. The absorbance of crystal violet was determined, andthe percent cell viability was graphed, using the absorbance of thenon-radiated mock-infected cells as 100% cell viability. As illustratedin FIG. 16, an approximately linear decrease in cell viability in allwells was obtained with increasing radiation dose, although the slope ofthe line was more negative with KD1, KD3, GZ1, or GZ3 than with mock,dl01/07, or dl309. With KD1, KD3, GZ1, and GZ3, there was much more celllysis and vector spread with their parental control viruses, and therewas synergy between the vectors and radiation. For example, withmock-infected cells, 600 cGy reduced cell viability by about 30% (70% ofcells were viable). KD1 without radiation reduced cell viability byabout 23%. The combination of 600 cGy radiation plus KD1 reduced cellviability to about 85%, more than 53% of which is the sum of radiationalone and KD1 alone. When considering the data in FIGS. 15 and 16together, a dose of about 600 cGy is optimal in this type of cellculture experiment.

The combination of KD3 or GZ3 with radiation was also examined in theA549 tumor-nude mouse model (see Example 4). A549 cells were injectedinto the hind flanks of nude mice, and tumors were allowed to form. Whentumors reached approximately 50-μl, they were injected with buffer orwith 5×10⁸ PFU of KD3 or GZ3. Eight to ten tumors were injected per testcondition. At 1 day p.i., half the mice received 600 cGy of whole bodyradiation. Tumor size was measured over time, and was plotted as afold-increase in tumor size versus days p.i. (as described in Example4). As shown in FIG. 17, the non-radiated buffer-injected tumors grewfaster than those injected with KD3 or GZ3. Tumors that received thecombination of KD3 and radiation did not grow, and those that receivedthe combination of GZ3 and radiation shrank in size after 14 days. Theseresults indicate that the combination of KD3 plus radiation or GZ3 plusradiation is more effective than either vector alone or radiation alonein reducing the rate of A549 tumor growth in nude mice. It is likelythat radiation would increase the effectiveness in treating tumors ofKD1 and GZ1, or indeed any other replication-competent orreplication-defective Ad vector.

The mechanism by which radiation causes the ADP overexpressing vectorsto lyse cells and spread from cell-to-cell more effectively is notunderstood. Radiation is expected to induce cellular DNA repairmechanisms, and that may allow for more efficient synthesis of Ad DNA.Radiation may enhance the function of ADP. ADP probably functions byinteracting with one or more cellular proteins, and radiation may affectthis protein(s) such that ADP functions more efficiently.

It is believed that KD1, KD3, GZ1, or GZ3, or any otherreplication-competent Ad vector, when used in combination withradiation, will be more effective than vector alone or radiation alonein providing clinical benefit to patients with cancer. The vectorsshould allow more tumor destruction with a given amount of radiation.Stated another way, radiation should cause more tumor destruction with agiven amount of vector. These vectors should also allow the radiationoncologist to use less radiation to achieve the same amount of tumordestruction. Less radiation would reduce the side effects of theradiation.

It is also believed that a cocktail of vectors when used in combinationwith radiation will be more effective than the cocktail alone orradiation alone. The cocktail could consist of ADP producing vectorsplus one or more replication defective vectors expressing an anticancertherapeutic protein (see Example 5).

EXAMPLE 9

This example illustrates a structure-function analysis of adenovirusdeath protein.

ADP is an 11.6 kDa N-linked O-linked integral membrane glycoprotein thatlocalizes to the inner nuclear membrane (NM) (Scaria et al., Virology191:743-753). As illustrated in FIG. 18, the Ad2-encoded ADP (SEQ IDNO:6) consists of 101 amino acids; aa 1-40 (SEQ ID NO:17) are lumenal,aa 41-59 (SEQ ID NO:18) constitute the transmembrane signal-anchor (SA)domain, aa 63-70 (SEQ ID NO:19) constitute a basic proline (BP) domainwithin the nucleoplasmic (NP) domain, which constitutes aa 61-101 (SEQID NO:20). To determine which domains in ADP are required to promotecell death, a number of deletion mutants of rec700 were prepared whichlacked various portions of the ADP gene and examined for the ability ofADP to localize to the NM and promote death. The rec700 virus is anAd5-Ad-Ad5 recombinant which has been described elsewhere (Wold et al.,Virology 148:168-180, 1986).

The structure of ADP in rec700 and in each deletion mutant isschematically illustrated in FIG. 18. The ADP gene in each deletionmutant has been sequenced using PCR methods to insure that the mutationsare correct. The structure and activity of ADP in the deletion mutantswas tested by infecting A549 cells followed by immunoblot analysis ofthe ADP mutant proteins as well as the ability to lyse cells. Alldeletion mutants expressed a stable ADP protein except pm734.1 (Δ1-48,i.e. aa 1-48 are deleted). The pm734.7 (N₁₄) ADP, which has Asn₁₄mutated to Ser, is O-glycosylated but not N-glycosylated because Asn₁₄is the only N-glycosylation site (data not shown). The dl735 (Δ4-11) ADPis N-glycosylated but not O-glycosylated because the sites forO-glycosylation are deleted (data not shown). The pm734.4 (M56) ADP,which has Met₅₆ in the SA domain mutated to Ser, contains exclusivelyN-linked high-mannose oligosaccharides (data not shown); this occursbecause the Met₅₆ mutation precludes exit of ADP from the endoplasmicreticulum (ER). The dl738 ADP, which lacks aa 46-60 in the signal-anchordomain, forms insoluble aggregates in the cytoplasm; therefore, aa 41-59do in fact include the signal-anchor domain. The pm734 (Δ1-40) ADP,which initiates at Met₄₁ at the N-terminus of the SA domain, comigratedwith the lower group of bands generated by proteolytic processing (datanot shown). This indicates that the proteolytic cleavage sites occurnear Met₄₁. Consistent with this, the proteolytic products were not seenwith dl737 (Δ29-45) (data not shown). Also, the size of the productsdecreased in all mutants with deletions within aa 41-101 (dl715.1,dl715, dl714, dl716) (data not shown).

The ability of these mutants to promote cell death was monitored bytrypan blue exclusion, plaque development, and lactate dehydrogenaserelease assays (Tollefson et al., J. Virol. 70:2296-2306, 1996). Thetrypan blue results in FIG. 15A indicate that the death-promotingfunction of ADP was abolished by deletion of aa 1-40 (pm734), aa 11-26(dl736.1), aa 18-22 (dl735.1), or aa 4-11 (dl735). Mutation of theN-glycosylation site at Asn₁₄ (pm734.7) reduced the death-promotingactivity to about 50% of rec700 (WT). dl737 (Δ29-45) was efficient asrec700 in promoting cell death; this indicates that the proteolyticprocessing products must not be required to promote cell death becausethey are not formed with dl737. The SA domain is essential for deathbecause dl738 (Δ46-60) and pm734.4 (M56) were completely defective (FIG.19). dl715.1 was nearly completely defective, indicating that the BPdomain is extremely important. Surprisingly, aa 71-94 (dl714), 76-89(dl715), and 79-101 (dl716) could be deleted without affecting thedeath-promoting activity of ADP (FIG. 19). On the other hand, deletionof aa 81-88 (dl717) nearly completely abolished the activity of ADP(FIG. 19); this is probably the result of aberrant sorting of ADP (seebelow). Similar results were obtained when the ability of these ADPmutants to promote cell death was examined with standard plaquedevelopment, LDH-release and MTT assays.

The effects of these mutations on the intracellular localization of ADPare extremely interesting. When examined by immunofluorescence (IF) at33 h p.i. (data not shown), ADP from rec700 (WT) localized crisply tothe NM; localization to the Golgi was also apparent. With dl714 (Δ71-94)and dl715 (Δ76-89), ADP localized to all membranes, i.e. the ER, Golgi,plasma membrane, and NM. This was even more apparent at 45 h p.i. (datanot shown) Thus, aa 71-94 appear to include a signal that directs ADPspecifically to the NM. ADP is very likely sorted from the trans-Golginetwork (TGN) to the NM, so this putative signal in ADP probablyfunctions in this sorting pathway. ADP from dl717 (Δ81-88) isintriguing: it localized to the NM and Golgi, but in many cells “dots”and circular structures were observed. Again, this was more apparent at45 h p.i. when these structures were the prominent feature.dl717-infected cells have not begun to die at 45 h p.i., so thesestructures are not cellular remnants. The intriguing possibility is thatthese structures are membrane vesicles that have pinched off from theTGN but are defective in targeting to and/or fusing with the NM.

With dl738 (Δ46-60 in the SA domain), ADP aggregated in the cytoplasm.This again indicates that aa 46-60 include the SA sequence. With pm734.4(M56), ADP localized primarily to the NM. As discussed above, thepm734.4 ADP has exclusively high-mannose N-linked oligosaccharides,indicating that it never leaves the ER. Perhaps the putativeNM-localization signal in the C-terminal region of the pm734.4 ADPtargets ADP to the NM by lateral diffusion from the ER (which iscontinuous with the outer and inner NM).

With dl737 (Δ29-45), ADP localized to the NM. ADP from pm734 (Δ1-40),pm734.7 (N14) (N-linked glycosylation cannot occur), and dl735 (Δ4-11;the O-glycosylation sites are deleted) localized much more prominentlyto the Golgi than the NM. ADP from dl735.1 (Δ18-22) and dl736.1 (Δ11-26)also localized much more strongly to the Golgi than the NM. Thus,residues 1-26 and/or glycosylation appear to be required for efficienttransport of ADP from the Golgi/TGN to the NM.

In summary, aa 41-59 include the SA domain, Met₅₆ in the SA domain isrequired for exit from the ER, aa 1-26 are required for efficient exitfrom the Golgi, and aa 76-94 are required to target ADP specifically tothe NM. With respect to promoting cell death, the essential regions areaa 1-26, the SA domain (ADP does not enter membranes), Met₅₆ in the SAdomain, and the BP domain (aa 63-70). It is not clear whether thedefective death-promoting phenotype of pm734 (Δ1-40), dl735 (Δ4-11),dl735.1 (Δ18-22), dl736.1 (Δ11-26), and pm734.7 (N14) is due to lack ofsequences (or oligosaccharides) that promote death or to much slowerexit of ADP from the Golgi to the NM. dl714 (Δ71-94) and dl715 (Δ76-89)express a wild-type phenotype for promoting death even though they aredefective in localizing specifically to the NM; this is probably becausesufficient ADP still enters the NM to promote death. Even though thedeletion in dl717 (Δ81-88) lies within the deletions in dl715 (Δ76-89)and dl714 (Δ71-94), the dl717 ADP is only about 15% as efficient asrec700 (WT), dl715 and dl714 in promoting death. This may be because thedl717 ADP tends to remain in vesicles rather than localizing to the NM.Altogether, these data indicate that ADP must localize to the NM inorder to promote cell death.

In view of the above, it will be seen that the several advantages of theinvention are achieved and other advantageous results attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

All references cited in this specification, including patents and patentapplications, are hereby incorporated by reference. The discussion ofreferences herein is intended merely to summarize the assertions made bytheir authors and no admission is made that any reference constitutesprior art. Applicants reserve the right to challenge the accuracy andpertinency of the cited references.

1. A method for treating cancer in an animal having a tumor comprisingadministering to the tumor an adenovirus vector wherein said adenovirusvector is replication-competent in a normal cell and a tumor cell andoverexpresses an adenovirus death protein (ADP), wherein overexpressionis defined as overexpression relative to dl309.
 2. The method of claim1, wherein overexpression relative to dl309 is detectable by westernblot, cell lysis, virus release or by a cell spreading assay.
 3. Themethod of claim 2, wherein the overexpression relative to dl309 isdetectable by western blot.
 4. The method of claim 2, wherein theoverexpression relative to dl309 is detectable by cell lysis.
 5. Themethod of claim 2, wherein the overexpression relative to dl309 isdetectable by virus release.
 6. The method of claim 2, wherein theoverexpression relative to dl309 is detectable by a cell spreadingassay.
 7. The method of claim 1, wherein the adenovirus vector comprisesSEQ ID NO:3 or SEQ ID NO:4.
 8. The method of claim 1, wherein theadenovirus death protein comprises SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ IDNO:12.
 9. The method of claim 1, further comprising the step ofpassively immunizing the animal against the adenovirus vector.
 10. Themethod of claim 1, further comprising treating the tumor with radiation.11. The method of claim 10, further comprising administering arecombinant adenovirus that is replication-defective to the tumor andtreating the tumor with radiation.
 12. The method of claim 1, furthercomprising treating the tumor with chemotherapy.
 13. The method of claim1, further comprising administering to the tumor one or morereplication-defective adenoviruses, wherein each replication-defectiveadenovirus expresses an anti-cancer gene product, and wherein theadenovirus vector facilitates the spread of the replication-defectiveadenovirus in the tumor.
 14. The method of claim 1, wherein theadenovirus vector lacks expression of at least one E3 protein selectedfrom the group consisting of gp19K, RIDα, RIDβ and 14.7K.
 15. The methodof claim 14, wherein the adenovirus vector lacks expression of the gp19Kprotein.
 16. The method of claim 14, wherein the adenovirus vector lacksexpression of the RIDα protein.
 17. The method of claim 14, wherein theadenovirus vector lacks expression of the RIDβ protein.
 18. The methodof claim 14, wherein the adenovirus vector lacks expression of the 14.7Kprotein.
 19. The method of claim 14, wherein the adenovirus vector lacksexpression of the gp19K, RIDα, RIDβ and 14.7K proteins.
 20. The methodof claim 1, wherein the adenovirus vector comprises a deletion in the E3region that removes a splice site for any of the E3 mRNAs.
 21. Themethod of claim 1, wherein the adenovirus vector comprises at least onedeletion in the E3 region, wherein the at least one deletion comprises asequence that encodes at least one E3 protein, wherein the protein isselected from the group consisting of gp19K, RIDα, RIDβ, and 14.7K. 22.The method of claim 21, wherein the at least one deletion comprises asequence that encodes the gp19K, RIDα, RIDβ and 14.7K proteins.
 23. Themethod of claim 22, wherein the at least one deletion further comprisesa sequence that encodes the 6.7K protein.
 24. The method of claim 22,wherein the at least one deletion further comprises a sequence thatencodes the 12.5K protein.
 25. The method of claim 22, wherein the atleast one deletion further comprises a sequence that encodes the 6.7Kand 12.5K proteins.
 26. The method of claim 1, wherein the animal is ahuman.
 27. The method of claim 1, wherein: a) the ADP is expressed froman ADP coding sequence positioned under the control of a promoter otherthan the endogenous promoters for ADP; b) the adenovirus vectorcomprises a deletion in the E3 region that removes a splice site for anE3 mRNA; c) the ADP is expressed from an ADP coding sequence flanked bya pre-mRNA splicing and cleavage/polyadenylation signal other than thepre-mRNA splicing and cleavage/polyadenylation signal normallyassociated with the ADP gene, or d) the ADP is expressed from an ADPcoding sequence that is positioned downstream of the coding sequence foranother adenovirus mRNA, together with a sequence on the 5′ side of theADP coding sequence that allows for internal initiation of translationof ADP.
 28. The method of claim 1, wherein the ADP is expressed from anADP coding sequence positioned under the control of promoter other thanthe endogenous promoters for ADP.
 29. The method of claim 28, whereinthe ADP coding sequence is positioned under the control of a promoterthat is exogenous to adenovirus.
 30. The method of claim 27, wherein theanimal is a human.
 31. The method of claim 1, wherein the ADP codingsequence is positioned behind a coding sequence for another adenovirusmRNA together with a sequence on the 5′ side of the ADP coding sequencethat allows for internal initiation of translation of ADP.
 32. Themethod of claim 31, wherein the sequence on the 5′ side of the ADPcoding sequence that allows for internal initiation of translation ofADP is an Ad tripartite leader or a viral internal ribosome initiationsequence.
 33. The method of claim 19, wherein the adenovirus vectoradditionally lacks expression of the E3 6.7K and 12.5K proteins.
 34. Themethod of claim 1, wherein the adenovirus vector is an Ad1, Ad2, Ad5 orAd6 vector.
 35. The method of claim 1, wherein the adenovirus vector isadministered to the tumor by injection of vector intravenously orintrathecally.
 36. The method of claim 1, wherein the adenovirus vectoris administered to the tumor by direct injection of the tumor.
 37. Themethod of claim 27, wherein the animal is passively immunized againstthe recombinant adenovirus.